| 1. |
Anderson GL,
Williams J,
Hille R,
( 1992 ) The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase. PMID : 1331097 : Abstract >>
The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.
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2. |
Braker G,
Tiedje JM,
( 2003 ) Nitric oxide reductase (norB) genes from pure cultures and environmental samples. PMID : 12788753 : DOI : 10.1128/aem.69.6.3476-3483.2003 PMC : PMC161466 Abstract >>
A PCR-based approach was developed to recover nitric oxide (NO) reductase (norB) genes as a functional marker gene for denitrifying bacteria. norB database sequences grouped in two very distinct branches. One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome bc-type complex (cNorB). The latter oxidizes cytochrome c, and the gene is localized adjacent to norC. While both norB types occur in denitrifying strains, the qnorB type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO. Branch-specific degenerate primer sets detected the two norB types in our denitrifier cultures. Specificity was confirmed by sequence analysis of the norB amplicons and failure to amplify norB from nondenitrifying strains. These primer sets also specifically amplified norB from freshwater and marine sediments. Pairwise comparison of amplified norB sequences indicated minimum levels of amino acid identity of 43.9% for qnorB and 38% for cnorB. Phylogenetic analysis confirmed the existence of two classes of norB genes, which clustered according to the respective primer set. Within the qnorB cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster. Most environmental qnorB clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms. cnorB clones were located on separate branches within subclusters of genes from known organisms, suggesting an origin from similar organisms.
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3. |
Liaw SH,
Chen SJ,
Ko TP,
Hsu CS,
Chen CJ,
Wang AH,
Tsai YC,
( 2003 ) Crystal structure of D-aminoacylase from Alcaligenes faecalis DA1. A novel subset of amidohydrolases and insights into the enzyme mechanism. PMID : 12454005 : DOI : 10.1074/jbc.M210795200 Abstract >>
D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the hydrolysis of N-acyl-D-amino acids. We report here the first D-aminoacylase crystal structure from A. faecalis at 1.5-A resolution. The protein comprises a small beta-barrel, and a catalytic (betaalpha)(8)-barrel with a 63-residue insertion. The enzyme structure shares significant similarity to the alpha/beta-barrel amidohydrolase superfamily, in which the beta-strands in both barrels superimpose well. Unexpectedly, the enzyme binds two zinc ions with widely different affinities, although only the tightly bound zinc ion is required for activity. One zinc ion is coordinated by Cys(96), His(220), and His(250), while the other is loosely chelated by His(67), His(69), and Cys(96). This is the first example of the metal ion coordination by a cysteine residue in the superfamily. Therefore, D-aminoacylase defines a novel subset and is a mononuclear zinc metalloenzyme but containing a binuclear active site. The preferred substrate was modeled into a hydrophobic pocket, revealing the substrate specificity and enzyme catalysis. The 63-residue insertion containing substrate-interacting residues may act as a gate controlling access to the active site, revealing that the substrate binding would induce a closed conformation to sequester the catalysis from solvent.
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4. |
Hsu CS,
Lai WL,
Chang WW,
Liaw SH,
Tsai YC,
( 2002 ) Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1. PMID : 12381838 : DOI : 10.1110/ps.0220902 PMC : PMC2373723 Abstract >>
D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the zinc-assistant hydrolysis of N-acyl-D-amino acids. We report here the cloning, expression, and structural-based mutation of the D-aminoacylase from Alcaligenes faecalis DA1. A 1,007-bp PCR product amplified with degenerate primers, was used to isolate a 4-kb genomic fragment, encoding a 484-residue D-aminoacylase. The enzyme amino-terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D-PSSM to be similar to urease and dihydroorotase, which have grouped into a novel alpha/beta-barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His-67, His-69, and His-250, putative metal ligands in D-aminoacylase, have been mutated previously, the remaining histidine (His-220) and aspartate (Asp-366) Asp-65, and four cysteines were then characterized. Substitution of Asp-65, Cys-96, His-220, and Asp-366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc-binding ability, revealing that Cys-96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino-terminal segment including Asp-65 may be responsible for structural stabilization.
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5. |
Chistoserdov AY,
( 2001 ) Cloning, sequencing and mutagenesis of the genes for aromatic amine dehydrogenase from Alcaligenes faecalis and evolution of amine dehydrogenases. PMID : 11495996 : DOI : 10.1099/00221287-147-8-2195 Abstract >>
The nucleotide sequence of the aromatic amine utilization (aau) gene region from Alcaligenes faecalis contained nine genes (orf-1, aauBEDA, orf-2, orf-3, orf-4 and hemE) transcribed in the same direction. The aauB and aauA genes encode the periplasmic aromatic amine dehydrogenase (AADH) large and small subunit polypeptides, respectively, and were homologous to mauB and mauA, the genes for the large and small subunits of methylamine dehydrogenase (MADH). aauE and aauD are homologous to mauE and mauD and apparently carry out the same function of transport and folding of the small subunit polypeptide in the periplasm. No analogues of the mauF, mauG, mauL, mauM and mauN genes responsible for biosynthesis of tryptophan tryptophylquinone (the prosthetic group of amine dehydrogenases) were found in the aau cluster. orf-2 was predicted to encode a small periplasmic monohaem c-type cytochrome. No biological function can be assigned to polypeptides encoded by orf-1, orf-3 and orf-4 and mutations in these genes appeared to be lethal. Mutants generated by insertions into mauD were not able to use phenylethylamine, tyramine and tryptamine as a source of carbon and phenylethylamine, 3'-hydroxytyramine (dopamine) and tyramine as a source of nitrogen, indicating that AADH is the only enzyme involved in utilization of primary amines in A. faecalis. AADH genes are present in Alcaligenes xylosoxydans subsp. xylosoxydans, but not in other beta- and gamma-proteobacteria. Phylogenetic analysis of amine dehydrogenases (MADH and AADH) indicated that AADH and MADH evolutionarily diverged before separation of proteobacteria into existing subclasses.
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6. |
Ellis PJ,
Conrads T,
Hille R,
Kuhn P,
( 2001 ) Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 A and 2.03 A. PMID : 11250197 : Abstract >>
Arsenite oxidase from Alcaligenes faecalis NCIB 8687 is a molybdenum/iron protein involved in the detoxification of arsenic. It is induced by the presence of AsO(2-) (arsenite) and functions to oxidize As(III)O(2-), which binds to essential sulfhydryl groups of proteins and dithiols, to the relatively less toxic As(V)O(4)(3-) (arsenate) prior to methylation. Using a combination of multiple isomorphous replacement with anomalous scattering (MIRAS) and multiple-wavelength anomalous dispersion (MAD) methods, the crystal structure of arsenite oxidase was determined to 2.03 A in a P2(1) crystal form with two molecules in the asymmetric unit and to 1.64 A in a P1 crystal form with four molecules in the asymmetric unit. Arsenite oxidase consists of a large subunit of 825 residues and a small subunit of approximately 134 residues. The large subunit contains a Mo site, consisting of a Mo atom bound to two pterin cofactors, and a [3Fe-4S] cluster. The small subunit contains a Rieske-type [2Fe-2S] site. The large subunit of arsenite oxidase is similar to other members of the dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes, particularly the dissimilatory periplasmic nitrate reductase from Desulfovibrio desulfuricans, but is unique in having no covalent bond between the polypeptide and the Mo atom. The small subunit has no counterpart among known Mo protein structures but is homologous to the Rieske [2Fe-2S] protein domain of the cytochrome bc(1) and cytochrome b(6)f complexes and to the Rieske domain of naphthalene 1,2-dioxygenase.
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7. |
Nishiyama M,
Tanokura M,
Kukimoto M,
( 2000 ) Gene organization for nitric oxide reduction in Alcaligenes faecalis S-6. PMID : 10830505 : Abstract >>
norB and norC encoding the cytochrome b-containing subunit and the cytochrome c-containing subunit, respectively, of the nitric oxide reductase (NOR) in Alcaligenes faecalis S-6 were cloned and sequenced. Both NorB and NorC showed more than 40% sequence identity to the corresponding subunits of cytochrome bc-type NORs in other denitrifying bacteria. norCB was in a gene cluster containing seven other genes; these were named dnr, orf2, orf3, norE, norF, norQ, and norD on the basis of their similarity with NOR systems in other bacteria. Potential FNR-binding sites were present in front of norCB, norEF, and/or orf2/orf3, suggesting that most of these genes are regulated simultaneously by an FNR-related protein. NorB and NorC proteins produced in the membrane fraction in Escherichia coli showed no enzyme activity, probably due to lack of NorQ and NorD, which appear to perform some essential function for activation of the NorB-NorC complex in the recombinant E. coli.
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8. |
Uchida Y,
Kobayashi M,
Goto M,
Hatakeyama K,
( 2000 ) Molecular analysis of maleate cis-trans isomerase from thermophilic bacteria. PMID : 10803955 : DOI : 10.1271/bbb.64.569 Abstract >>
Several strains of thermophilic bacteria containing maleate cis-trans isomerase were isolated from soil samples and identified as Bacillus stearothermophilus, Bacillus circulans, Bacillus brevis, and Deleya halophila. The maleate cis-trans isomerase was purified and characterized from one of the isolated strains, B. stearothermophilus MI-102. The purified enzyme of strain MI-102 showed higher thermal stability than the enzyme of a mesophile, Alcaligenes faecalis IFO13111. The seven maleate cis-trans isomerase genes (maiA) of thermophile were cloned and sequenced. B. stearothemophilus MI-102 MaiA has 67% amino acid identity with A. faecalis MaiA. All eight amino acid sequences of maiA gene products had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. To probe the catalytic mechanism, three cysteine residues in the conserved regions of A. faecalis MaiA were replaced with serine by site-directed mutagenesis. The results suggest that Cys80 and Cys198 play important roles in the enzyme activity.
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9. |
Sun D,
Zhu Z,
( 1999 ) Localization of periplasmic redox proteins of Alcaligenes faecalis by a modified general method for fractionating gram-negative bacteria. PMID : 10515948 : PMC : PMC103793 Abstract >>
A lysozyme-osmotic shock method is described for fractionation of Alcaligenes faecalis which uses glucose to adjust osmotic strength and multiple osmotic shocks. During phenylethylamine-dependent growth, aromatic amine dehydrogenase, azurin, and a single cytochrome c were localized in the periplasm. Their induction patterns are different from those for the related quinoprotein methylamine dehydrogenase and its associated redox proteins.
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10. |
Hallin S,
Lindgren PE,
( 1999 ) PCR detection of genes encoding nitrite reductase in denitrifying bacteria. PMID : 10103263 : PMC : PMC91233 Abstract >>
Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.
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11. |
Sukumar N,
Chen ZW,
Ferrari D,
Merli A,
Rossi GL,
Bellamy HD,
Chistoserdov A,
Davidson VL,
Mathews FS,
( 2006 ) Crystal structure of an electron transfer complex between aromatic amine dehydrogenase and azurin from Alcaligenes faecalis. PMID : 17087503 : DOI : 10.1021/bi0612972 Abstract >>
The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of the TTQ-containing methylamine dehydrogenase (MADH) and the cupredoxin amicyanin. Despite significant similarities between the two quinoproteins and the two cupredoxins, each is specific for its respective partner and the ionic strength dependence and magnitude of the binding constant for each complex are quite different. The AADH-azurin interface is largely hydrophobic, covering approximately 500 A(2) of surface on each molecule, with one direct hydrogen bond linking them. The closest distance from TTQ to copper is 12.6 A compared with a distance of 9.3 A in the MADH-amicyanin complex. When the MADH-amicyanin complex is aligned with the AADH-azurin complex, the amicyanin lies on top of the azurin but is oriented quite differently. Although the copper atoms differ in position by approximately 4.7 A, the amicyanin bound to MADH appears to be rotated approximately 90 degrees from its aligned position with azurin. Comparison of the structures of the two complexes identifies features of the interface that dictate the specificity of the protein-protein interaction and determine the rate of interprotein electron transfer.
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12. |
Roujeinikova A,
Scrutton NS,
Leys D,
( 2006 ) Atomic level insight into the oxidative half-reaction of aromatic amine dehydrogenase. PMID : 17005560 : DOI : 10.1074/jbc.M605559200 Abstract >>
The quinoprotein aromatic amine dehydrogenase (AADH) uses a covalently bound tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. Recent crystal structures have provided insight into the reductive half-reaction. In contrast, no atomic details are available for the oxidative half-reaction. The TTQ O7 hydroxyl group is protonated during reduction, but it is unclear how this proton can be removed during the oxidative half-reaction. Furthermore, compared with the electron transfer from the N-quinol form, electron transfer from the non-physiological O-quinol form to azurin is significantly slower. Here we report crystal structures of the O-quinol, N-quinol, and N-semiquinone forms of AADH. A comparison of oxidized and substrate reduced AADH species reveals changes in the TTQ-containing subunit, extending from residues in the immediate vicinity of the N-quinol to the putative azurin docking site, suggesting a mechanism whereby TTQ redox state influences interprotein electron transfer. In contrast, chemical reduction of the TTQ center has no significant effect on protein conformation. Furthermore, structural reorganization upon substrate reduction places a water molecule near TTQ O7 where it can act as proton acceptor. The structure of the N-semiquinone, however, is essentially similar to oxidized AADH. Surprisingly, in the presence of substrate a covalent N-semiquinone substrate adduct is observed. To our knowledge this is the first detailed insight into a complex, branching mechanism of quinone oxidation where significant structural reorganization upon reduction of the quinone center directly influences formation of the electron transfer complex and nature of the electron transfer process.
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13. |
Masgrau L,
Roujeinikova A,
Johannissen LO,
Hothi P,
Basran J,
Ranaghan KE,
Mulholland AJ,
Sutcliffe MJ,
Scrutton NS,
Leys D,
( 2006 ) Atomic description of an enzyme reaction dominated by proton tunneling. PMID : 16614214 : DOI : 10.1126/science.1126002 Abstract >>
We present an atomic-level description of the reaction chemistry of an enzyme-catalyzed reaction dominated by proton tunneling. By solving structures of reaction intermediates at near-atomic resolution, we have identified the reaction pathway for tryptamine oxidation by aromatic amine dehydrogenase. Combining experiment and computer simulation, we show proton transfer occurs predominantly to oxygen O2 of Asp(128)beta in a reaction dominated by tunneling over approximately 0.6 angstroms. The role of long-range coupled motions in promoting tunneling is controversial. We show that, in this enzyme system, tunneling is promoted by a short-range motion modulating proton-acceptor distance and no long-range coupled motion is required.
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14. |
Agersø Y,
Sandvang D,
( 2005 ) Class 1 integrons and tetracycline resistance genes in alcaligenes, arthrobacter, and Pseudomonas spp. isolated from pigsties and manured soil. PMID : 16332771 : DOI : 10.1128/AEM.71.12.7941-7947.2005 PMC : PMC1317413 Abstract >>
The presence of tetracycline resistance (Tc(r)) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc(r) gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli, Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc(r) genes tet(A), tet(B), and tet(C), and all (n = 72) gram-positive isolates were screened for tet(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet(B). All gram-positive isolates with in-1 also contained tet(33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc(r) and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc(r) but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.
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15. |
Hothi P,
Khadra KA,
Combe JP,
Leys D,
Scrutton NS,
( 2005 ) Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis. Cofactor assembly and catalytic properties of recombinant enzyme expressed in Paracoccus denitrificans. PMID : 16279953 : DOI : 10.1111/j.1742-4658.2005.04990.x Abstract >>
The heterologous expression of tryptophan trytophylquinone (TTQ)-dependent aromatic amine dehydrogenase (AADH) has been achieved in Paracoccus denitrificans. The aauBEDA genes and orf-2 from the aromatic amine utilization (aau) gene cluster of Alcaligenes faecalis were placed under the regulatory control of the mauF promoter from P. denitrificans and introduced into P. denitrificans using a broad-host-range vector. The physical, spectroscopic and kinetic properties of the recombinant AADH were indistinguishable from those of the native enzyme isolated from A. faecalis. TTQ biogenesis in recombinant AADH is functional despite the lack of analogues in the cloned aau gene cluster for mauF, mauG, mauL, mauM and mauN that are found in the methylamine utilization (mau) gene cluster of a number of methylotrophic organisms. Steady-state reaction profiles for recombinant AADH as a function of substrate concentration differed between 'fast' (tryptamine) and 'slow' (benzylamine) substrates, owing to a lack of inhibition by benzylamine at high substrate concentrations. A deflated and temperature-dependent kinetic isotope effect indicated that C-H/C-D bond breakage is only partially rate-limiting in steady-state reactions with benzylamine. Stopped-flow studies of the reductive half-reaction of recombinant AADH with benzylamine demonstrated that the KIE is elevated over the value observed in steady-state turnover and is independent of temperature, consistent with (a) previously reported studies with native AADH and (b) breakage of the substrate C-H bond by quantum mechanical tunnelling. The limiting rate constant (k(lim)) for TTQ reduction is controlled by a single ionization with pK(a) value of 6.0, with maximum activity realized in the alkaline region. Two kinetically influential ionizations were identified in plots of k(lim)/K(d) of pK(a) values 7.1 and 9.3, again with the maximum value realized in the alkaline region. The potential origin of these kinetically influential ionizations is discussed.
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16. |
Mantengoli E,
Rossolini GM,
( 2005 ) Tn5393d, a complex Tn5393 derivative carrying the PER-1 extended-spectrum beta-lactamase gene and other resistance determinants. PMID : 16048938 : DOI : 10.1128/AAC.49.8.3289-3296.2005 PMC : PMC1196282 Abstract >>
In Alcaligenes faecalis FL-424/98, a clinical isolate that produces the PER-1 extended-spectrum beta-lactamase, the bla(PER-1) gene was found to be carried on a 44-kb nonconjugative plasmid, named pFL424, that was transferred to Escherichia coli by electroporation. Investigation of the genetic context of the bla(PER-1) gene in pFL424 by means of a combined cloning and PCR mapping approach revealed that the gene is associated with a transposonlike element of the Tn3 family. This 14-kb element is a Tn5393 derivative of original structure, named Tn5393d, which contains the transposition module and the strAB genes typical of other members of the Tn5393 lineage plus additional resistance determinants, including the bla(PER-1) gene and a new allelic variant of the aphA6 aminoglycoside phosphotransferase gene, named aphA6b, whose product is active against kanamycin, streptomycin, and amikacin. Tn5393d apparently originated from the consecutive insertion of two composite transposons into a Tn5393 backbone carrying the aphA6b and the bla(PER-1) genes, respectively. The putative composite transposon carrying bla(PER-1), named Tn4176, is made of two original and nonidentical insertion sequences of the IS4 family, named IS1387a and IS1387b, of which one is interrupted by the insertion of an original insertion sequence of the IS30 family, named IS1066. In pFL424, Tn5393d is inserted into a Tn501-like mercury resistance transposon. Transposition of Tn5393d or modules thereof containing the bla(PER-1) gene from pFL424 to small multicopy plasmids or to a bacterial artificial chromosome was not detected in an E. coli host harboring both replicons.
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17. |
Wilson MM,
Metcalf WW,
( 2005 ) Genetic diversity and horizontal transfer of genes involved in oxidation of reduced phosphorus compounds by Alcaligenes faecalis WM2072. PMID : 15640200 : DOI : 10.1128/AEM.71.1.290-296.2005 PMC : PMC544259 Abstract >>
Enrichment was performed to isolate organisms that could utilize reduced phosphorus compounds as their sole phosphorus sources. One isolate that grew well with either hypophosphite or phosphite was identified by 16S rRNA gene analysis as a strain of Alcaligenes faecalis. The genes required for oxidation of hypophosphite and phosphite by this organism were identified by using transposon mutagenesis and include homologs of the ptxD and htxA genes of Pseudomonas stutzeri WM88, which encode an NAD-dependent phosphite dehydrogenase (PtxD) and 2-oxoglutarate-dependent hypophosphite dioxygenase (HtxA). This organism also has the htxB, htxC, and htxD genes that comprise an ABC-type transporter, presumably for hypophosphite and phosphite transport. The role of these genes in reduced phosphorus metabolism was confirmed by analyzing the growth of mutants in which these genes were deleted. Sequencing data showed that htxA, htxB, htxC, and htxD are virtually identical to their homologs in P. stutzeri at the DNA level, indicating that horizontal gene transfer occurred. However, A. faecalis ptxD is very different from its P. stutzeri homolog and represents a new ptxD lineage. Therefore, this gene has ancient evolutionary roots in bacteria. These data suggest that there is strong evolutionary selection for the ability of microorganisms to oxidize hypophosphite and phosphite.
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18. |
Lai WL,
Chou LY,
Ting CY,
Kirby R,
Tsai YC,
Wang AH,
Liaw SH,
( 2004 ) The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation. PMID : 14736882 : DOI : 10.1074/jbc.M308849200 Abstract >>
Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1 belongs to the beta-mononuclear subset due to the fact that the catalytically essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96), His(220), and His(250), even though it possesses a binuclear active site with a weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+), Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96), and Asp(366), revealing that elimination of the beta site changes the coordination geometry of the alpha ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the d-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the beta site, but inhibited by the subsequent binding of the second ion at the alpha site.
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19. |
Curson AR,
Sullivan MJ,
Todd JD,
Johnston AW,
( 2011 ) DddY, a periplasmic dimethylsulfoniopropionate lyase found in taxonomically diverse species of Proteobacteria. PMID : 21248856 : PMC : PMC3146280 DOI : 10.1038/ismej.2010.203 Abstract >>
The abundant compatible solute dimethylsulfoniopropionate (DMSP) is made by many marine algae. Different marine bacteria catabolise DMSP by various mechanisms, some of which liberate the environmentally important gas dimethyl sulfide (DMS). We describe an enzyme, DddY, which cleaves DMSP into DMS plus acrylate and is located in the bacterial periplasm, unlike other DMSP lyases that catalyse this reaction. There are dddY-like genes in strains of Alcaligenes, Arcobacter and Shewanella, in the �]-, ?- and �^-proteobacteria, respectively. In Alcaligenes, dddY is in a cluster of ddd and acu genes that resemble, but also have significant differences to, those in other bacteria that catabolise both DMSP and acrylate. Although production of DMS and transcription of Alcaligenes dddY are both apparently inducible by pre-growth of cells with DMSP, this substrate must be catabolised to form acrylate, the bona fide coinducer.
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20. |
Hoque MM,
Shimizu S,
Juan EC,
Sato Y,
Hossain MT,
Yamamoto T,
Imamura S,
Suzuki K,
Amano H,
Sekiguchi T,
Tsunoda M,
Takénaka A,
( 2009 ) Structure of D-3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate D-3-hydroxybutyrate and NAD+. PMID : 19342772 : DOI : 10.1107/S1744309109008537 PMC : PMC2664752 Abstract >>
D-3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between D-3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate D-3-hydroxybutyrate and the cofactor NAD(+) at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD(+) and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of D-3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure.
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21. |
Hoque MM,
Shimizu S,
Hossain MT,
Yamamoto T,
Imamura S,
Suzuki K,
Tsunoda M,
Amano H,
Sekiguchi T,
Takénaka A,
( 2008 ) The structures of Alcaligenes faecalis D-3-hydroxybutyrate dehydrogenase before and after NAD+ and acetate binding suggest a dynamical reaction mechanism as a member of the SDR family. PMID : 18453685 : DOI : 10.1107/S0907444908004009 Abstract >>
D-3-Hydroxybutyrate dehydrogenase, which catalyzes the reversible reaction between D-3-hydroxybutyrate and acetoacetate, has been classified into the short-chain dehydrogenase/reductase family and is a useful marker in the assay of diabetes mellitus and/or ketoacidosis. The enzyme from Alcaligenes faecalis was crystallized in the apo form and in the holo form with acetate as a substrate analogue. The crystal structures of both forms were determined at 2.2 angstroms resolution. The enzyme is a tetramer composed of four subunits assembled with noncrystallographic 222 point symmetry. Each subunit has two domains. The principal domain adopts the Rossmann fold essential for nucleotide binding, which is a common feature of the SDR family. NAD+ is bound in a large cleft in the domain. The pyrophosphate group of NAD+ is covered by the small additional domain, which is supported by two extended arms allowing domain movement. In the catalytic site, a water molecule is trapped by the catalytic Tyr155 and Ser142 residues in the vicinity of the bound NAD+ and acetate. The substrate analogue acetate is bound above the nicotinamide plane. A substrate (D-3-hydroxybutylate) bound model can reasonably be constructed by adding two C atoms into the void space between the water O atom and the methyl group of the acetate, suggesting a substrate-bound state before enzymatic reaction occurs. Based on these structural features, a reaction mechanism has been proposed.
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22. |
Yamamoto K,
Uozumi T,
Beppu T,
( 1987 ) The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue copper protein from Escherichia coli cells. PMID : 2824441 : DOI : 10.1128/jb.169.12.5648-5652.1987 PMC : PMC214024 Abstract >>
The gene encoding a blue copper protein (a member of the pseudoazurins) of 123 amino acid residues, containing a single type I Cu2+ ion, was cloned from Alcaligenes faecalis S-6. The nucleotide sequence of the coding region, as well as the 5'- and 3'-flanking regions, was determined. The deduced amino acid sequence after Glu-24 coincided with the reported sequence of the blue protein, and its NH2-terminal sequence of 23 residues resembled a typical signal peptide. The cloned gene was expressed under the control of the tac promoter in Escherichia coli, and the correctly processed blue protein was secreted into the periplasm. The blue protein produced in E. coli possessed the activity to transfer electrons to the copper-containing nitrite reductase of A. faecalis S-6 in vitro.
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23. |
Qiu J,
Zhang J,
Zhang Y,
Wang Y,
Tong L,
Hong Q,
He J,
( 2017 ) Biodegradation of Picolinic Acid by a Newly Isolated Bacterium Alcaligenes faecalis Strain JQ135. PMID : 28243718 : DOI : 10.1007/s00284-017-1205-2 Abstract >>
We isolated a bacterial strain JQ135 from municipal wastewater, which was capable of efficiently degrading picolinic acid (PA). Based on the physico-biochemical characteristics and 16S rDNA analysis, strain JQ135 was identified as Alcaligenes faecalis. In addition, strain JQ135 produced an orange pigment when cultured in the Luria-Bertani medium, which is different from the previously reported strains of A. faecalis. During the degradation of PA by the resting strain JQ135 cells, only one intermediate, 6-hydroxypicolinic acid (6HPA), was detected by ultraviolet spectrophotometry, high-pressure liquid chromatography, and liquid chromatography-mass spectrometry. A random transposon mutagenesis library of strain JQ135 was constructed. One mutant, Mut-G31, could convert PA into 6HPA without further degradation. The disrupted gene (orf2) was amplified from Mut-G31, and its product showed 32% identity to the 3-deoxy-D-manno-octulosonic acid kinase (KdkA) from Haemophilus influenzae. Results from complementation analysis confirmed that GTG was the initiation codon of the kdkA-like orf2, and that it was essential for PA biodegradation by strain JQ135. This study provides the first genetic evidence for the bacterial degradation of PA.
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24. |
Paiva MC,
Reis MP,
Costa PS,
Dias MF,
Bleicher L,
Scholte LLS,
Nardi RMD,
Nascimento AMA,
( 2017 ) Identification of new bacteria harboring qnrS and aac(6')-Ib/cr and mutations possibly involved in fluoroquinolone resistance in raw sewage and activated sludge samples from a full-scale WWTP. PMID : 27984803 : DOI : 10.1016/j.watres.2016.11.056 Abstract >>
Wastewater treatment plants (WWTPs) harbor bacteria and antimicrobial resistance genes, favoring gene exchange events and resistance dissemination. Here, a culture-based and metagenomic survey of qnrA, qnrB, qnrS, and aac(6')-Ib genes from raw sewage (RS) and activated sludge (AS) of a full-scale municipal WWTP was performed. A total of 96 bacterial isolates were recovered from nalidixic acid-enrichment cultures. Bacteria harboring the aac(6')-Ib gene predominated in RS, whereas qnrS-positive isolates were specific to AS. Novel qnrS- and aac(6')-Ib-cr positive species were identified: Morganella morganii, Providencia rettgeri, and Pseudomonas guangdongensis (qnrS), and Alcaligenes faecalis and P. rettgeri (aac(6')-Ib-cr). Analysis of qnrS and aac(6')-Ib sequences from isolates and clone libraries suggested that the diversity of qnrS is wider than that of aac(6')-Ib. A large number of amino acid mutations were observed in the QnrS and AAC(6')-Ib proteins at previously undetected positions, whose structural implications are not clear. An accumulation of mutations at the C72, Q73, L74, A75 and M76 positions of QnrS, and D181 of AAC(6')-Ib might be important for resistance. These findings add significant information on bacteria harboring qnrS and aac(6')-Ib genes, and the presence of novel mutations that may eventually emerge in clinical isolates.
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25. |
Mishra P,
Kaur S,
Sharma AN,
Jolly RS,
( 2016 ) Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid. PMID : 27391673 : DOI : 10.1371/journal.pone.0159009 PMC : PMC4938524 Abstract >>
Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S)-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R)-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S)-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline)-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S)-amide to (S)-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH). IaaH is known to catalyse conversion of indole-3-acetamide (IAM) to indole-3-acetic acid (IAA), which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To the best of our knowledge this is the first application of IaaH in production of industrially important molecules.
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26. |
Kanazawa H,
Hoque MM,
Tsunoda M,
Suzuki K,
Yamamoto T,
Kawai G,
Kondo J,
Takénaka A,
( 2016 ) Structural insights into the catalytic reaction trigger and inhibition of D-3-hydroxybutyrate dehydrogenase. PMID : 27380367 : DOI : 10.1107/S2053230X16007767 PMC : PMC4933000 Abstract >>
D-3-Hydroxybutyrate dehydrogenase catalyzes the reversible conversion of acetoacetate and D-3-hydroxybutyrate. These ketone bodies are both energy-storage forms of acetyl-CoA. In order to clarify the structural mechanisms of the catalytic reaction with the cognate substrate D-3-hydroxybutyrate and of the inhibition of the reaction by inhibitors, the enzyme from Alcaligenes faecalis has been analyzed by X-ray crystallography in liganded states with the substrate and with two types of inhibitor: malonate and methylmalonate. In each subunit of the tetrameric enzyme, the substrate is trapped on the nicotinamide plane of the bound NAD(+). An OMIT map definitively shows that the bound ligand is D-3-hydroxybutyrate and not acetoacetate. The two carboxylate O atoms form four hydrogen bonds to four conserved amino-acid residues. The methyl group is accommodated in the nearby hydrophobic pocket so that the formation of a hydrogen bond from the OH group of the substrate to the hydroxy group of Tyr155 at the active centre is facilitated. In this geometry, the H atom attached to the C(3) atom of the substrate in the sp(3) configuration is positioned at a distance of 3.1 ? from the nicotinamide C(4) atom in the direction normal to the plane. In addition, the donor-acceptor relationship of the hydrogen bonds suggests that the Tyr155 OH group is allowed to ionize by the two donations from the Ser142 OH group and the ribose OH group. A comparison of the protein structures with and without ligands indicates that the Gln196 residue of the small movable domain participates in the formation of additional hydrogen bonds. It is likely that this situation can facilitate H-atom movements as the trigger of the catalytic reaction. In the complexes with inhibitors, however, their principal carboxylate groups interact with the enzyme in a similar way, while the interactions of other groups are changed. The crucial determinant for inhibition is that the inhibitors have no active H atom at C(3). A second determinant is the Tyr155 OH group, which is perturbed by the inhibitors to donate its H atom for hydrogen-bond formation, losing its nucleophilicity.
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27. |
Mauger J,
Yamada H,
( 1990 ) A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3. Purification and characterization. PMID : 2269298 : DOI : 10.1111/j.1432-1033.1990.tb19467.x Abstract >>
A new type of nitrilase, arylacetonitrilase, has been purified from isovaleronitrile-induced cells of Alcaligenes faecalis JM3 in four steps. The purity of the enzyme was confirmed by SDS/polyacrylamide gel electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 275 kDa and consists of six subunits of identical molecular mass. The purified enzyme exhibits a pH optimum of 7.5 and a temperature optimum of 45 degrees C. The enzyme is specific for arylacetonitriles such as 2-thiophenacetonitrile, p-tolyacetonitrile, p-chlorobenzylcyanide, p-fluorobenzylcyanide and 3-pyridylacetonitrile. The enzyme stoichiometrically catalyzes the hydrolysis of arylacetonitrile to arylacetic acid and ammonia, no formation of amide occurring. However, the enzyme does not attack nitrile groups attached to aromatic and heteroaromatic rings, which are hydrolyzed preferably by the nitrilases known previously. The enzyme requires thiol compounds such as dithiothreitol and 2-mercaptoethanol to exhibit its maximum activity.
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28. |
Liu ZQ,
Dong LZ,
Cheng F,
Xue YP,
Wang YS,
Ding JN,
Zheng YG,
Shen YC,
( 2011 ) Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10. PMID : 21913706 : DOI : 10.1021/jf202746a Abstract >>
Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 �XC, respectively. Mg(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+), Ni(2+), Ag(+), and Hg(2+) showed a strong inhibitory effect. The K(m) and v(max) for mandelonitrile were 4.74 mM and 15.85 �gmol min(-1) mg(-1) protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.
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29. |
Lett MC,
Muller D,
Lièvremont D,
Silver S,
Santini J,
( 2012 ) Unified nomenclature for genes involved in prokaryotic aerobic arsenite oxidation. PMID : 22056935 : DOI : 10.1128/JB.06391-11 PMC : PMC3256664 Abstract >>
N/A
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30. |
( 1997 ) Domains of Escherichia coli acyl carrier protein important for membrane-derived-oligosaccharide biosynthesis. PMID : 9171419 : DOI : 10.1128/jb.179.11.3697-3705.1997 PMC : PMC179167 Abstract >>
Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate. By contrast, the phosphopantetheine prosthetic group is not required for membrane-derived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis. These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51. The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36. Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesized by the mechanism found in E. coli.
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31. |
( 1997 ) Gene cloning and characterization of maleate cis-trans isomerase from Alcaligenes faecalis. PMID : 9345272 : DOI : 10.1006/bbrc.1997.7430 Abstract >>
Maleate cis-trans isomerase, which catalyses the conversion of maleate to fumarate, was purified and characterized from Alcaligenes faecalis IFO13111. The molecular weight of maleate isomerase was estimated as 60 kDa, consisting of a 28 kDa dimer as shown by gel-filtration chromatography and SDS-PAGE analysis. Kinetic studies showed that the Michaelis constant for maleate was 4.0 x 10(-5) M. The reverse reaction (fumarate to maleate) activity of the enzyme was detected even though it was quite weak. The maleate isomerase gene (maiA) was cloned by hybridization using the oligonucleotide DNA probes designed on the basis of the determined N-terminal amino acid sequences of the purified enzyme. The determined DNA sequence of the maiA gene contains an open reading frame which encodes a 254-amino-acid sequence. The amino acid sequence of the maiA gene product shows no significant homology to any amino acid sequences in the protein data base.
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32. |
( 1997 ) Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis. PMID : 9292993 : PMC : PMC168649 Abstract >>
Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.
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33. |
( 1997 ) Structure of nitrite bound to copper-containing nitrite reductase from Alcaligenes faecalis. Mechanistic implications. PMID : 9353305 : DOI : 10.1074/jbc.272.45.28455 Abstract >>
The structures of oxidized, reduced, nitrite-soaked oxidized and nitrite-soaked reduced nitrite reductase from Alcaligenes faecalis have been determined at 1.8-2.0 A resolution using data collected at -160 degrees C. The active site at cryogenic temperature, as at room temperature, contains a tetrahedral type II copper site liganded by three histidines and a water molecule. The solvent site is empty when crystals are reduced with ascorbate. A fully occupied oxygen-coordinate nitrite occupies the solvent site in crystals soaked in nitrite. Ascorbate-reduced crystals soaked in a glycerol-methanol solution and nitrite at -40 degrees C remain colorless at -160 degrees C but turn amber-brown when warmed, suggesting that NO is released. Nitrite is found at one-half occupancy. Five new solvent sites in the oxidized nitrite bound form exhibit defined but different occupancies in the other three forms. These results support a previously proposed mechanism by which nitrite is bound primarily by a single oxygen atom that is protonable, and after reduction and cleavage of that N-O bond, NO is released leaving the oxygen atom bound to the Cu site as hydroxide or water.
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34. |
( 1993 ) Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues. PMID : 8419930 : DOI : 10.1073/pnas.90.1.247 PMC : PMC45637 Abstract >>
Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence deduced from the nucleotide sequence of the nitrilase gene shows 34.7% identity with that of Klebsiella ozaenae nitrilase. A DNA clone containing the nitrilase gene expressed the active enzyme in Escherichia coli with excellent yield. Among five cysteine residues (Cys-40, Cys-115, Cys-162, Cys-163, and Cys-218) in the Alcaligenes nitrilase, only Cys-163 was conserved at the corresponding position in the Klebsiella nitrilase. Two mutant enzymes, in which Cys-162 and Cys-163 were replaced with Asn and Ala, respectively, were constructed by site-directed mutagenesis. A 35% increase of the specific activity and a large reduction of the Km for thiophene-2-acetonitrile (which was used as a standard substrate for the nitrilase) were observed in the Cys-162-->Asn mutant enzyme. The Cys-163-->Ala mutation resulted in complete loss of nitrilase activity, clearly indicating that Cys-163 is crucial for the activity and Cys-162 could not provide the catalytic function of Cys-163.
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35. |
( 1994 ) Aromatic amine dehydrogenase, a second tryptophan tryptophylquinone enzyme. PMID : 8188594 : DOI : 10.1128/jb.176.10.2922-2929.1994 PMC : PMC205448 Abstract >>
Aromatic amine dehydrogenase (AADH) catalyzes the oxidative deamination of aromatic amines including tyramine and dopamine. AADH is structurally similar to methylamine dehydrogenase (MADH) and possesses the same tryptophan tryptophylquinone (TTQ) prosthetic group. AADH exhibits an alpha 2 beta 2 structure with subunit molecular weights of 39,000 and 18,000 and with a quinone covalently attached to each beta subunit. Neither subunit cross-reacted immunologically with antibodies to the corresponding subunits of MADH, and the N-terminal amino acid sequence of the beta subunit of AADH exhibited no homology with the highly conserved beta subunits of MADH. The absorption spectra for the oxidized, semiquinone, and reduced forms of AADH have been characterized, and extinction coefficients for the absorption maxima of each redox form have been determined. These spectra are very similar to those for MADH, indicating the likelihood of a TTQ cofactor. This was verified by the near identity of the vibrational frequencies and intensities in the resonance Raman spectra for the oxidized forms of AADH and MADH. A stable semiquinone of AADH could be observed during a reductive titration with dithionite, whereas titration with tyramine proceeded directly from the oxidized to the reduced form. AADH was very stable against denaturation by heat and exposure to guanidine. The individual subunits could be separated by gel filtration after incubation in guanidine hydrochloride, and partial reconstitution of activity was observed on recombination of the subunits. Steady-state kinetic analysis of AADH yielded a Vmax of 17 mumol/min/mg and a Km for tyramine of 5.4 microM. Substrate inhibition by tyramine was observed. AADH was irreversibly inhibited by hydrazine, phenylhydrazine, hydroxylamine, semicarbazide, and aminoguanidine. Isonicotinic acid hydrazide (isoniazid) and isonicotinic acid 2-isopropyl hydrazide (iproniazid) were reversible noncompetitive inhibitors of AADH and exhibited K(i) values of 8 and 186 microM, respectively. The similarities and differences between AADH and other amine oxidizing enzymes are also discussed.
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36. |
( 1993 ) Cloning and characterization of a nitrite reductase gene from Alcaligenes faecalis and its expression in Escherichia coli. PMID : 8515232 : DOI : 10.1099/00221287-139-4-725 Abstract >>
The gene (nir) encoding the copper-containing nitrite reductase (NIR) of a denitrifying bacterium, Alcaligenes faecalis S-6, was cloned by a synthetic oligonucleotide-probing method. The nucleotide sequence of the cloned DNA fragment revealed the primary structure of the NIR precursor containing the N-terminal signal sequence for secretion. A nucleotide sequence, possibly recognized by a transcriptional regulator resembling FNR was found upstream of the structural gene. When the cloned gene was expressed in Escherichia coli under the control of the lac promoter at 37 degrees C, NIR was produced as large inclusion bodies and little activity was detected. When cultivation was at 20 degrees C, most of the NIR was detected in the soluble fraction and a significant portion of the protein was translocated into the periplasmic space, accompanied by removal of its signal sequence.
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37. |
( 1994 ) X-ray structure and site-directed mutagenesis of a nitrite reductase from Alcaligenes faecalis S-6: roles of two copper atoms in nitrite reduction. PMID : 8172899 : DOI : 10.1021/bi00183a030 Abstract >>
Nitrite reductase (NIR) from the denitrifying bacterium Alcaligenes faecalis S-6 is a copper-containing enzyme which requires pseudoazurin, a low molecular weight protein containing a single type I copper atom, as a direct electron donor in vivo. Crystallographic analysis shows that NIR is a trimer composed of three identical subunits, each of which contains one atom of type I copper and one atom of type II copper, and that the ligands to the type I and type II copper atoms are the same as those of the Achromobacter cycloclastes NIR. An efficient NIR expression-secretion system in Escherichia coli was constructed and used for site-directed mutagenesis. An NIR mutant with a replacement of the type II copper ligand, His135, by Lys still retained a type II copper site as well as a type I copper atom, but it completely lost nitrite-reducing activity as measured with methyl viologen as an electron donor. On the other hand, another mutant with a replacement of the type I copper ligand, Met150, by Glu contained only a type II copper atom, but it still retained significant nitrite-reducing activity with methyl viologen. When pseudoazurin was used as an electron donor for the reaction, however, Met150Glu failed to catalyze the reduction of nitrite. Kinetic analysis of the electron transfer between NIR and pseudoazurin revealed that the electron-transfer rate between Met150Glu and pseudoazurin was reduced 1000-fold relative to that of wild-type NIR.(ABSTRACT TRUNCATED AT 250 WORDS)
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38. |
Engeseth HR,
McMillin DR,
Otvos JD,
( 1984 ) Comparative Cd-113 nuclear magnetic resonance studies of Cd(II)-substituted blue copper proteins. PMID : 6232270 : Abstract >>
The 113Cd NMR spectra of plastocyanin (Spinacea), stellacyanin (Rhus vernicifera), and two azurins (Pseudomonas aeruginosa and Alcaligenes faecalis) have been measured after introducing Cd(II) into the blue copper-binding sites. Relative to Cd(C1O4)2 the chemical shifts are 432, 380, 372, and 379 ppm, respectively, all of which are found to be reasonable values for binding sites containing a cysteine thiolate ligand. The 113Cd resonances of the cadmium derivatives of stellacyanin and the azurins are so near the same that the proteins must present very similar metal-binding sites. In contrast the plastocyanin derivative resonates about 50 ppm further downfield which may signal a change in coordination number. The spin lattice relaxation times of the 113Cd resonances are of the order of 0.1 s, and a major portion of the relaxation apparently occurs through the chemical shift anisotropy mechanism. At 13 degrees C the 113Cd resonance of Psuedomonas azurin shifts slightly downfield with increasing pH. This is explained by a small change in the environment about cadmium which occurs as a result of the conformational change that attends the titration of His-35.
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39. |
Petratos K,
Banner DW,
Beppu T,
Wilson KS,
Tsernoglou D,
( 1987 ) The crystal structure of pseudoazurin from Alcaligenes faecalis S-6 determined at 2.9 A resolution. PMID : 3595868 : DOI : 10.1016/0014-5793(87)81048-7 Abstract >>
The three-dimensional structure of pseudoazurin, a single copper-containing protein from Alcaligenes faecalis strain S-6, has been determined at 2.9 A resolution by X-ray crystallography. The sequences of two other pseudoazurins from Pseudomonas AM1 and Achromobacter cycloclastes may also be accommodated in this structure. The structure, an eight-stranded beta-barrel, resembles closely those of plastocyanin and azurin. It possesses two extra alpha-helices at the C-terminus, whereas azurins have an alpha-helical flap in the middle of their sequences.
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40. |
Murphy ME,
Turley S,
Kukimoto M,
Nishiyama M,
Horinouchi S,
Sasaki H,
Tanokura M,
Adman ET,
( 1995 ) Structure of Alcaligenes faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc. PMID : 7547950 : DOI : 10.1021/bi00038a003 Abstract >>
The structures at 2.0 and 2.25 A resolution of native and recombinant nitrite reductase from Alcaligenes faecalis show that they are identical to each other and very similar to nitrite reductase from Achromobacter cycloclastes. The crystallographic structure of a mutant, M150E, which unlike the wild-type protein cannot be reduced by pseudoazurin, shows that the glutamate replacement for methionine binds to a metal at the type I Cu site via only one oxygen. Anomalous scattering data collected at wavelengths of 1.040 and 1.377 A reveal that the metal at the type I site is a Zn. No significant differences from the native structure other than local perturbations at the type I site are seen. A local pseudo 2-fold axis relates the two domains of different monomers which form the active site. The two residues, Asp98 and His255, believed to be involved in catalysis are related by this 2-fold. An unusual (+)-(+) charge interaction between Lys269, Glu279, and His100 helps to orient the active site Cu ligand, His100. A number of negatively charged surface residues create an electrostatic field whose shape suggests that it may serve to direct incoming negatively charged nitrite as well as to dock the electron donor partner, pseudoazurin.
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41. |
Edwards SL,
Davidson VL,
Hyun YL,
Wingfield PT,
( 1995 ) Spectroscopic evidence for a common electron transfer pathway for two tryptophan tryptophylquinone enzymes. PMID : 7876189 : DOI : 10.1074/jbc.270.9.4293 Abstract >>
Aromatic amine dehydrogenase (AADH) and methylamine dehydrogenase (MADH) are the only two enzymes known to use the cofactor tryptophan tryptophylquinone (TTQ). Each catalyzes oxidative deamination of a distinct class of primary amines. A detailed comparison of their circular dichroic spectra indicates that both proteins share a similar fold with their TTQ cofactors residing in similar environments and that this may be a useful diagnostic probe for TTQ enzymes. Alcaligenes faecalis cells induced to express AADH also express a large amount of the blue copper protein, azurin. Oxidized azurin is rapidly reduced by a catalytic amount of AADH in the presence of the substrate, tyramine. Three A. faecalis cytochromes-c and three other cytochromes-c were tested for electron transfer activity with AADH. Azurin markedly facilitated electron transfer from AADH to each cytochrome. This suggests that AADH and azurin may form an electron transfer complex with a c-type cytochrome, analogous to the crystallographically determined MADH-amicyanin-cytochrome c-551i complex (Chen, L., Durley, R. C. E., Matthews, F. S., and Davidson, V. L. (1994) Science 264, 86-90). The similarities of MADH and AADH plus the demonstration of azurin and multiple cytochromes as functional electron-transfer partners suggest that both TTQ-bearing enzymes share common mechanisms for oxidative deamination and subsequent electron transfer.
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42. |
Rosen P,
Segal M,
Pecht I,
( 1981 ) Electron transfer between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa. PMID : 6274637 : DOI : 10.1111/j.1432-1033.1981.tb05709.x Abstract >>
The electron transfer equilibrium and kinetics between azurin from Alcaligenes faecalis and cytochrome c551 from Pseudomonas aeruginosa have been studied. The equilibrium constant K = ([Cyt(III)] . [Az(I)])/([Cyt(II)] . [Az(II))]) = 0.5 at 25 degrees C is about seven times smaller than that observed between the cytochrome c551 and the titrations confirmed a 43-mV difference between the mid-point potentials of +266 mV and +309 mV for the Alcaligenes and Pseudomonas azurins respectively. The kinetics of the reaction between Alcaligenes azurin and Pseudomonas cytochrome c551 were investigated by the temperature-jump chemical relaxation method. Only a single relaxation mode was observed throughout the range of concentrations and temperatures examined. Thus, the slow relaxation time observed in the reaction between P. aeruginosa azurin and cytochrome c551 is not observed with the Alcaligenes azurin. The simplest mechanism that can therefore be ascribed to the investigated system is: [formula: see text]. This scheme is similar to that proposed earlier for the reaction between P. aeruginosa azurin and cytochrome c551 but does not involve the conformational transition proposed for azurin. The specific rates for the electron transfer are still fast: 1.8 x 10(6) M-1 . s-1 and 3.0 x 10(6) M-1 . s-1 respectively at 25 degrees C.
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43. |
Mitra S,
Bersohn R,
( 1982 ) Proton NMR of the histidines of azurin from Alcaligenes faecalis: linkage of histidine-35 with redox kinetics. PMID : 6960351 : DOI : 10.1073/pnas.79.22.6807 PMC : PMC347222 Abstract >>
On the basis of redox kinetic studies, Rosen and Pecht [Rosen, P. & Pecht, I. (1976) Biochemistry 15, 775-786] postulated a slowly attained (approximately equal to 0.1 sec) conformational equilibrium between two forms of reduced azurin from the bacterium Pseudomonas aeruginosa, one form being faster in electron transfer. NMR investigations have shown that at pH 7 there are two forms of reduced azurin exchanging slowly with each other, differing in the presence or absence of a proton on the imidazole side chain of histidine-35. Rosen et al. [Rosen, P., Segal, M. & Pecht, I. (1981) Eur. J. Biochem. 120, 339-344] observed that the azurin from the bacterium Alcaligenes faecalis shows no such slowly attained equilibrium between two forms. Therefore, a 1H NMR study was carried out on this azurin with emphasis on the downfield region. A resonance was found at 7.95 ppm downfield that does not move with pH, is not seen in the oxidized protein, has the same pseudocontact shift in the Co(II) derivative as the C-2 proton of histidine-35 has in the Co(II) derivative of P. aeruginosa azurin, and, in the apoprotein, exhibits a typical protonation shift downfield at pH less than 5. Therefore, this resonance is assigned to the C-2 proton of histidine-35. The crystal structure of P. aeruginosa azurin shows that at pH 7 the imidazole side chain of histidine-35 is in a crevice within the protein, where its ring is adjacent and parallel to that of histidine-47, a copper ligand. The preceding observations combined with others show that the kinetics of some redox reactions involving azurin depend on the position of histidine-35. The implication is that there is a pathway for electron transport to the copper atom involving passage through histidine-35.
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44. |
Hormel S,
Adman E,
Walsh KA,
Beppu T,
Titani K,
( 1986 ) The amino acid sequence of the blue copper protein of Alcaligenes faecalis. PMID : 3512305 : DOI : 10.1016/0014-5793(86)80346-5 Abstract >>
The complete amino acid sequence of a blue copper protein from Alcaligenes faecalis S-6 has been determined. This protein is clearly homologous to pseudoazurins in Achromobacter cycloclastes and Pseudomonas AM1, more distantly related to plant plastocyanins, and markedly different from the azurin of Pseudomonas aeruginosa. Yet all of these proteins bind copper, and analogous ligands appear to be involved.
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45. |
Adman ET,
Turley S,
Bramson R,
Petratos K,
Banner D,
Tsernoglou D,
Beppu T,
Watanabe H,
( 1989 ) A 2.0-A structure of the blue copper protein (cupredoxin) from Alcaligenes faecalis S-6. PMID : 2909547 : Abstract >>
The structure of a blue copper protein, cupredoxin, from the potent denitrifying bacterium Alcaligenes faecalis S-6, has been determined and refined against 2 A x-ray diffraction data. The agreement between observed and calculated structure factors is 0.159, and estimated errors in coordinates are 0.09-0.15 A. The protein folds in a beta sandwich similar to plastocyanin and azurin and includes features such as a "kink" and a "tyrosine loop" which have been noted previously for these proteins as well as immunoglobulins. The copper is bound by four ligands, in a distorted tetrahedral arrangement, with Cu-S gamma = 2.07 A (Cys-78), Cu-N delta 1 = 2.10 and 2.21 for His-40 and His-81, and Cu-S delta = 2.69 A (Met-86). Two of the ligands are further oriented by hydrogen bonds either to other side chains (Asn-9 to His-40), backbone atoms (NH...S) or a water molecule (to His-40). The methionine ligand has no extra constraints. The C-terminal loop containing three of the ligands is hydrogen-bonded to the strand containing His-40 by hydrogen bonds between the conserved residues Thr-79 and Asn-41. The pronounced dichroism of the crystal is a result of the orientation of the normal to the C beta-S gamma-Cu plane parallel to the crystallographic 6-fold axis.
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46. |
Parks DH,
Chuvochina M,
Waite DW,
Rinke C,
Skarshewski A,
Chaumeil PA,
Hugenholtz P,
( 2018 ) A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. PMID : 30148503 : DOI : 10.1038/nbt.4229 Abstract >>
Taxonomy is an organizing principle of biology and is ideally based on evolutionary relationships among organisms. Development of a robust bacterial taxonomy has been hindered by an inability to obtain most bacteria in pure culture and, to a lesser extent, by the historical use of phenotypes to guide classification. Culture-independent sequencing technologies have matured sufficiently that a comprehensive genome-based taxonomy is now possible. We used a concatenated protein phylogeny as the basis for a bacterial taxonomy that conservatively removes polyphyletic groups and normalizes taxonomic ranks on the basis of relative evolutionary divergence. Under this approach, 58% of the 94,759 genomes comprising the Genome Taxonomy Database had changes to their existing taxonomy. This result includes the description of 99 phyla, including six major monophyletic units from the subdivision of the Proteobacteria, and amalgamation of the Candidate Phyla Radiation into a single phylum. Our taxonomy should enable improved classification of uncultured bacteria and provide a sound basis for ecological and evolutionary studies.
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47. |
Yalpani N,
Altier D,
Barry J,
Kassa A,
Nowatzki TM,
Sethi A,
Zhao JZ,
Diehn S,
Crane V,
Sandahl G,
Guan R,
Poland B,
Perez Ortega C,
Nelson ME,
Xie W,
Liu L,
Wu G,
( 2017 ) An Alcaligenes strain emulates Bacillus thuringiensis producing a binary protein that kills corn rootworm through a mechanism similar to Cry34Ab1/Cry35Ab1. PMID : 28596570 : DOI : 10.1038/s41598-017-03544-9 PMC : PMC5465095 Abstract >>
Crops expressing Bacillus thuringiensis (Bt)-derived insecticidal protein genes have been commercially available for over 15 years and are providing significant value to growers. However, there remains the need for alternative insecticidal actives due to emerging insect resistance to certain Bt proteins. A screen of bacterial strains led to the discovery of a two-component insecticidal protein named AfIP-1A/1B from an Alcaligenes faecalis strain. This protein shows selectivity against coleopteran insects including western corn rootworm (WCR). Transgenic maize plants expressing AfIP-1A/1B demonstrate strong protection from rootworm injury. Surprisingly, although little sequence similarity exists to known insecticidal proteins, efficacy tests using WCR populations resistant to two different Cry proteins show that AfIP-1A/1B and mCry3A differ in their mode of action while AfIP-1A/1B and the binary Cry34Ab1/Cry35Ab1 protein share a similar mode. These findings are supported by results of competitive binding assays and the similarity of the x-ray structure of AfIP-1A to Cry34Ab1. Our work indicates that insecticidal proteins obtained from a non-Bt bacterial source can be useful for developing genetically modified crops and can function similarly to familiar proteins from Bt.
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48. |
( 1998 ) Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. PMID : 9758798 : PMC : PMC106545 Abstract >>
A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.
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49. |
( 1998 ) Identification of reaction products and intermediates of aromatic-amine dehydrogenase by 15N and 13C NMR. PMID : 9494080 : DOI : 10.1042/bj3301159 PMC : PMC1219256 Abstract >>
13C- and 15N-NMR studies of the reaction of aromatic amine dehydrogenase (AADH) with methylamine demonstrated that the products of the reductive half-reaction are an equivalent of formaldehyde hydrate and a reduced aminoquinol form of the tryptophan tryptophylquinone (TTQ) cofactor which contains covalently bound substrate-derived N. These data are consistent with the Ping Pong kinetic mechanism and aminotransferase-type chemical reaction mechanism which have been previously proposed for AADH. Comparison of the 15N-NMR spectra of the aminoquinol TTQ intermediates of AADH and methylamine dehydrogenase (MADH) revealed that the substrate-derived aminoquinol N of AADH and MADH exhibited distinct 15N chemical shifts which are separated by approx. 7 p.p.m. In each case, the signal for the substrate-derived aminoquinol N appears optimally with short pulse delay and exhibits a relaxation time and chemical shift which are consistent with 15N covalently bound to an aromatic ring (i.e. aminoquinol) which is attached to a rigid protein matrix. The aminoquinol of AADH is less stable against reoxidation than that of MADH. These data suggest that differences in the active-site mediated electrostatic environments of the aminoquinol N in the respective enzymes may influence both the observed 15N chemical shift and the relative reactivities of the TTQ aminoquinols towards oxygen. These data also demonstrate the utility of 13C- and 15N-NMR spectroscopy as a tool for monitoring the intermediates and products of enzyme-catalysed transformations.
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