| 1. |
Kim Y,
Hol WG,
( 2001 ) Structure of cephalosporin acylase in complex with glutaryl-7-aminocephalosporanic acid and glutarate: insight into the basis of its substrate specificity. PMID : 11755403 : Abstract >>
Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), which is obtained by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be of great interest. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzyme has low turnover rates for CPC. The binary complex structures of CA with GL-7-ACA and glutarate (the side-chain of GL-7-ACA) show extensive interactions between the glutaryl moiety of GL-7-ACA and the seven residues that form the side-chain pocket. These interactions explain why the D-alpha-aminoadipyl side-chain of CPC yields a poorer substrate than GL-7-ACA. This understanding of the nature of substrate specificity may be useful in the design of an enzyme with an improved performance for the conversion of CPC to 7-ACA. Additionally, the catalytic mechanism of the deacylation reaction was revealed by the ligand bound structures.
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2. |
Kim Y,
Kim S,
Earnest TN,
Hol WG,
( 2002 ) Precursor structure of cephalosporin acylase. Insights into autoproteolytic activation in a new N-terminal hydrolase family. PMID : 11706000 : DOI : 10.1074/jbc.M108888200 Abstract >>
Autocatalytic proteolytic cleavage is a frequently observed post-translational modification in proteins. Cephalosporin acylase (CA) is a recently identified member of the N-terminal hydrolase family that is activated from an inactive precursor by autoproteolytic processing, generating a new N-terminal residue, which is either a Ser or a Thr. The N-terminal Ser or Thr becomes a nucleophilic catalytic center for intramolecular and intermolecular amide cleavages. The gene structure of the open reading frame of CAs generally consists of a signal peptide followed by the alpha-subunit, a spacer sequence, and the beta-subunit, which are all translated into a single polypeptide chain, the CA precursor. The precursor is post-translationally modified into an active heterodimeric enzyme with alpha- and beta-subunits, first by intramolecular cleavage and second by intermolecular cleavage. We solved the first CA precursor structure (code 1KEH) from a class I CA from Pseudomonas diminuta at a 2.5-A resolution that provides insight into the mechanism of intramolecular cleavage. A conserved water molecule, stabilized by four hydrogen bonds in unusual pseudotetrahedral geometry, plays a key role to assist the OG atom of Ser(1beta) to generate a strong nucleophile. In addition, the site of the secondary intermolecular cleavage of CA is proposed to be the carbonyl carbon of Gly(158alpha) (Kim, S., and Kim, Y., (2001) J. Biol. Chem., 276, 48376-48381), which is different from the situation in two other class I CAs.
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3. |
Han XY,
Andrade RA,
( 2005 ) Brevundimonas diminuta infections and its resistance to fluoroquinolones. PMID : 15883180 : DOI : 10.1093/jac/dki139 Abstract >>
To report infections caused by Brevundimonas diminuta and antibiotic studies of this Gram-negative bacterium. Seven patients with infection and eight bacterial strains were studied. Tests included antibiotic susceptibility and analysis of the DNA gyrase and topoisomerase genes and the effect of efflux pump inhibitor Phe-Arg-beta-naphthylamide (PANA). The patients all had underlying disease of cancer. The infections involved bloodstream (one case), intravascular catheter (four cases), urinary tract (one case) and pleural space (one case of empyema). Fever up to 39.2 degrees C characterized these infections, which resolved upon treatment by combination antibiotics. Microbiologically, all organisms were resistant to multiple fluoroquinolones and cefepime, but were susceptible to amikacin, imipenem and ticarcillin/clavulanate. These quinolone-resistant B. diminuta strains were probably selected out by the prophylactic use of a quinolone in six of these patients. Additionally, the B. diminuta type strain ATCC 11568(T) that was isolated before the quinolone era from water was also resistant to ciprofloxacin and intermediate to levofloxacin, suggesting intrinsic quinolone resistance. The DNA gyrase and topoisomerase of six analysed strains all contained GyrA Ala-83 and Met-87, GyrB Leu-466 or Thr-466, and ParC Gln-57, Val-66 and Ala-80 that were probably the cause of fluoroquinolone resistance. PANA had nearly negligible effect. B. diminuta is intrinsically resistant to fluoroquinolones and can be selected out to cause infections.
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4. |
Komeda H,
Hariyama N,
Asano Y,
( 2006 ) L: -Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family. PMID : 16001251 : DOI : 10.1007/s00253-005-0068-9 Abstract >>
Brevundimonas diminuta TPU 5720 produces an amidase acting L-stereoselectively on phenylalaninamide. The enzyme (LaaA(Bd)) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of approximately 53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50 degrees C and pH 7.5. LaaA(Bd) lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co(2+) ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co(2+), suggesting that LaaA(Bd) is a Co(2+)-dependent enzyme. LaaA(Bd) had hydrolyzing activity toward a broad range of L-amino acid amides including L-phenylalaninamide, L-glutaminamide, L-leucinamide, L-methioninamide, L-argininamide, and L-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA(Bd) was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA (Bd)) encodes the amidase. LaaA(Bd) is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA (Bd) gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA(Bd) in cell-free extracts of the E. coli transformant was 25.9 units mg(-1) with L-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.
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5. |
Hill JE,
Penny SL,
Crowell KG,
Goh SH,
Hemmingsen SM,
( 2004 ) cpnDB: a chaperonin sequence database. PMID : 15289485 : DOI : 10.1101/gr.2649204 PMC : PMC509277 Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
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6. |
Pandeeti EV,
Chakka D,
Pandey JP,
Siddavattam D,
( 2011 ) Indigenous organophosphate-degrading (opd) plasmid pCMS1 of Brevundimonasdiminuta is self-transmissible and plays a key role in horizontal mobility of the opd gene. PMID : 21354204 : DOI : 10.1016/j.plasmid.2011.02.003 Abstract >>
A fosmid library of the 66kb indigenous organophosphate-degrading (opd) plasmid pCMS1 of Brevundimonas diminuta was tagged with mini-transposon EZTn5
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7. |
Scotta C,
Bennasar A,
Moore ER,
Lalucat J,
Gomila M,
( 2011 ) Taxonomic characterisation of ceftazidime-resistant Brevundimonas isolates and description of Brevundimonas faecalis sp. nov. PMID : 21782367 : DOI : 10.1016/j.syapm.2011.06.001 Abstract >>
Three ceftazidime-resistant strains isolated from the sewage water of a municipal hospital in Palma de Mallorca, Spain, were analysed phenotypically and genotypically to clarify their taxonomic positions. Sequence determinations and phylogenetic analyses of the 16S rRNA genes indicated that strains CS20.3(T), CS39 and CS41 were affiliated with the species of the alphaproteobacterial genus Brevundimonas, most closely related to B. bullata, B. diminuta, B. naejangsanensis and B. terrae. Additional sequences analyses of the ITS1 region of the rRNA operon and the genes for the housekeeping enzymes DNA gyrase �]-subunit and RNA polymerase �]-subunit, genomic DNA-DNA hybridisation similarities, cell fatty acid profiles and physiological and biochemical characterizations supported the recognition of CS20.3(T) (CCUG 58127(T)=CECT 7729(T)) as a distinct and novel species, for which the name Brevundimonas faecalis sp. nov. is proposed. Strains CS39 and CS41 were ascribed to the species B. diminuta.
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8. |
Adelowo OO,
Fagade OE,
( 2009 ) The tetracycline resistance gene tet39 is present in both Gram-negative and Gram-positive bacteria from a polluted river, Southwestern Nigeria. PMID : 19196439 : DOI : 10.1111/j.1472-765X.2008.02523.x Abstract >>
Previous analysis of tet39 suggests it may be present in other bacterial species. Hence, we investigated the host range of tet39 among bacterial from a poultry waste polluted river in Southwestern Nigeria. Thirteen resistant bacterial isolated from the water and sediment of the polluted river was investigated for the presence of tetracycline resistance genes tetA, tetB, tetC, tet39 and the transposon integrase gene of the Tn916/1545 family by PCR. While tetA, tetB, tetC and integrase genes cannot be detected in any of the organisms, tet39 was detected in eight of the tested organisms including three Gram-positive species. Sequence analysis showed the genes have high sequence identities (> or =99%) with tet39 of Acinetobacter sp. LUH5605, the first and only bacterial genus from which the gene has been reported to date. This is a novel observation. This study shows that apart from Acinetobacter, tet39 is present in other bacterial species tested in this study. This study adds to available information on the occurrence and distribution of tet39 among environmental bacteria and suggests that the gene has a broader host range than previously reported.
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9. |
Tayeb LA,
Lefevre M,
Passet V,
Diancourt L,
Brisse S,
Grimont PA,
( 2008 ) Comparative phylogenies of Burkholderia, Ralstonia, Comamonas, Brevundimonas and related organisms derived from rpoB, gyrB and rrs gene sequences. PMID : 18280706 : DOI : 10.1016/j.resmic.2007.12.005 Abstract >>
Phylogenetic analysis of strains from Burkholderia, Ralstonia, Cupriavidus, Comamonas, Delftia, Acidovorax, Brevundimonas, Herbaspirillum huttiense and "Pseudomonas butanovora" was performed based on the protein-coding genes rpoB and gyrB and on the 16S rRNA-coding gene rrs. Overall, the phylogenies deduced from the three genes were concordant among themselves and with current taxonomy. However, a few differences among individual gene phylogenies were noted. For example, the separation of Cupriavidus from Ralstonia was not supported in the rpoB tree, as Ralstonia was nested within Cupriavidus. Similarly, the separation of Delftia from Comamonas was not supported in the gyrB tree. Based on rrs and rpoB, the genus Burkholderia contained four groups: (i) the B. cepacia complex, (ii) the B. pseudomallei-B. thailandensis group, (iii) a 6-species group including B. caledonica and B. glathei and (iv) the B. plantarii-B. glumae-B. gladioli group. However, B. caribensis and B. glathei stood as a fifth group based on gyrB. It appears that a phylogeny cannot be reliably based on a single gene. Using rpoB and gyrB, better separation of closely related species was obtained compared to rrs, indicating the potential of these two genes for identification and species definition. Nevertheless, intraspecific sequence diversity will need to be determined to fully establish the value of these genes for strain identification.
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10. |
McDaniel CS,
Harper LL,
Wild JR,
( 1988 ) Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase. PMID : 2834339 : DOI : 10.1128/jb.170.5.2306-2311.1988 PMC : PMC211123 Abstract >>
Plasmid pCMS1 was isolated from Pseudomonas diminuta MG, a strain which constitutively hydrolyzes a broad spectrum of organophosphorus compounds. The native plasmid was restricted with PstI, and individual DNA fragments were subcloned into pBR322. A recombinant plasmid transformed into Escherichia coli possessed weak hydrolytic activity, and Southern blotting with the native plasmid DNA verified that the DNA sequence originated from pCMS1. When the cloned 1.3-kilobase fragment was placed behind the lacZ' promoter of M13mp10 and retransformed into E. coli, clear-plaque isolates with correctly sized inserts exhibited isopropyl-beta-D-thiogalactopyranoside-inducible whole-cell activity. Sequence determination of the M13 constructions identified an open reading frame of 975 bases preceded by a putative ribosome-binding site appropriately positioned upstream of the first ATG codon in the open reading frame. An intragenic fusion of the opd gene with the lacZ gene produced a hybrid polypeptide which was purified by beta-galactosidase immunoaffinity chromatography and used to confirm the open reading frame of opd. The gene product, an organophosphorus phosphotriesterase, would have a molecular weight of 35,418 if the presumed start site is correct. Eighty to ninety percent of the enzymatic activity was associated with the pseudomonad membrane fractions. When dissociated by treatment with 0.1% Triton and 1 M NaCl, the enzymatic activity was associated with a molecular weight of approximately 65,000, suggesting that the active enzyme was dimeric.
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11. |
Campbell E,
Kaltenbach M,
Correy GJ,
Carr PD,
Porebski BT,
Livingstone EK,
Afriat-Jurnou L,
Buckle AM,
Weik M,
Hollfelder F,
Tokuriki N,
Jackson CJ,
( 2016 ) The role of protein dynamics in the evolution of new enzyme function. PMID : 27618189 : DOI : 10.1038/nchembio.2175 Abstract >>
Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.
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12. |
( 1997 ) Structural studies of malate dehydrogenases (MDHs): MDHs in Brevundimonas species are the first reported MDHs in Proteobacteria which resemble lactate dehydrogenases in primary structure. PMID : 9190829 : DOI : 10.1128/jb.179.12.4066-4070.1997 PMC : PMC179222 Abstract >>
The N-terminal sequences of malate dehydrogenases from 10 bacterial strains, representing seven genera of Proteobacteria, were determined. Of these, the enzyme sequences of species classified in the genus Brevundimonas clearly resembled those malate dehydrogenases with greatest similarity to lactate dehydrogenases. Additional evidence from subunit molecular weights, peptide mapping, and enzyme mobilities suggested that malate dehydrogenases from species of the genus Brevundimonas were structurally distinct from others in the study.
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13. |
( 1996 ) Three-dimensional structure of the zinc-containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzylphosphonate. PMID : 8634243 : DOI : 10.1021/bi960325l Abstract >>
Phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of paraoxon and related acetylcholinesterase inhibitors with rate enhancements that approach 10(12). The enzyme requires a binuclear metal center for activity and as isolated contains 2 equiv of zinc per subunit. Here we describe the three-dimensional structure of the Zn2+/Zn2+-substituted enzyme complexed with the substrate analog diethyl 4-methylbenzylphosphonate. Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.6 A, b = 91.4 A, c = 69.4 A, beta = 91.9 degrees, and two subunits in the asymmetric unit. The model was refined by least-squares analysis to a nominal resolution of 2.1 A and a crystallographic R-factor of 15.4% for all measured X-ray data. As in the previously reported structure of the cadmium-containing enzyme, the bridging ligands are a carbamylated lysine residue (Lys 169) and a hydroxide. The zinc ions are separated by 3.3 A. The more buried zinc ion is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging hydroxide in a trigonal bipyramidal arrangement as described for the cadmium-substituted enzyme. Unlike the octahedral coordination observed for the more solvent-exposed cadmium ion, however, the second zinc is tetrahedrally ligated to Lys 169, His 201, His 230, and the bridging hydroxide. The diethyl 4-methylbenzylphosphonate occupies a site near the binuclear metal center with the phosphoryl oxygen of the substrate analog situated at 3.5 A from the more solvent-exposed zinc ion. The aromatic portion of the inhibitor binds in a fairly hydrophobic pocket. A striking feature of the active site pocket is the lack of direct electrostatic interactions between the inhibitor and the protein. This most likely explains the broad substrate specificity exhibited by phosphotriesterase. The position of the inhibitor within the active site suggests that the nucleophile for the hydrolysis reaction is the metal-bound hydroxide.
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14. |
( 1994 ) Purification and characterization of isoquinoline 1-oxidoreductase from Pseudomonas diminuta 7, a novel molybdenum-containing hydroxylase. PMID : 8157655 : Abstract >>
Isoquinoline 1-oxidoreductase, which catalyzes the hydroxylation of isoquinoline to 1-oxo-1,2-dihydroisoquinoline with concomitant reduction of a suitable electron acceptor, was purified from the isoquinoline degrading bacterium Pseudomonas diminuta 7 to apparent homogeneity. The native enzyme was a heterodimer with a molecular mass of 95 kDa consisting of a 16- and a 80-kDa subunit. It contained 0.85 g atom molybdenum, 3.95 g atom iron, 3.9 g atom acid-labile sulfur, 2.1 mol of phosphate, and 1 mol of CMP/mol of enzyme. CMP and phosphate are suggested to originate from molybdopterin cytosine dinucleotide of the pterin molybdenum cofactor. It is assumed that the iron and the acid-labile sulfur are arranged in two (2Fe-2S) clusters. The isoelectric point of the isoquinoline 1-oxidoreductase was within the range of pH 6.2 to 6.8. Cytochrome c, ferricyanide, and several non-physiological electron acceptors served as oxidizing substrates, whereas O2 and NAD were not used. Isoquinoline 1-oxidoreductase revealed a high specificity toward the reducing substrates isoquinoline, 5-hydroxyisoquinoline, quinazoline, and phthalazine. Isoquinoline 1-oxidoreductase was inactivated by methanol, arsenite, p-hydroxymercuribenzoate, 1,10-phenanthroline, and cyanide. Additionally, the enzyme was inactivated upon incubation with its substrates isoquinoline, which slowly inhibited the enzyme in the absence of an electron acceptor, and 5-hydroxy-isoquinoline, which rapidly and very effectively inactivated the enzyme in the presence as well as in the absence of the electron acceptors iodonitrotetrazolium chloride, phenazine methosulfate, or ferricyanide.
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15. |
( 1994 ) Identification of the histidine ligands to the binuclear metal center of phosphotriesterase by site-directed mutagenesis. PMID : 8155644 : DOI : 10.1021/bi00180a022 Abstract >>
In order to identify which of the seven histidines in phosphotriesterase participate at the active site/binuclear metal center of the enzyme, site-directed mutagenesis has been employed to change, individually, each of the seven histidine residues to asparagine. In addition, the gene for the wild-type enzyme has been subcloned without its leader sequence behind a modified ribosomal binding site, leading to a 5-fold increase in protein expression. The seven mutants, H55N, H57N, H123N, H201N, H230N, H254N, and H257N, exhibit varying degrees of activity compared to the wild-type enzyme. The H123N and H257N mutants are as active as the wild-type enzyme, but all of the other mutant enzymes have 10% or less activity. The metal content of the cobalt-purified mutant enzymes has been determined to be less than that of the wild-type enzyme in all cases. Each of the mutant enzymes has been converted to apoenzyme and reconstituted with 2 equiv of zinc(II), cadmium(II), or cobalt(II). The kinetic parameters, Vmax and V/Km, and apparent pKa's have been determined for each of the reconstituted enzyme derivatives. In almost all cases, the apparent pKa's have shifted toward higher values. The pH-rate profiles for some of the reconstituted mutant enzymes are significantly different from those for the wild-type enzyme, indicating that other groups may become involved in the reaction mechanism upon mutation of the histidine residue to asparagine. His-123 is the only histidine residue that appears to have no involvement in the catalytic activity of phosphotriesterase.(ABSTRACT TRUNCATED AT 250 WORDS)
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16. |
( 1994 ) Three-dimensional structure of phosphotriesterase: an enzyme capable of detoxifying organophosphate nerve agents. PMID : 7999757 : DOI : 10.1021/bi00254a008 Abstract >>
Organophosphates, such as parathion and paraoxon, constitute the largest class of insecticides currently used in industrialized nations. In addition, many of these compounds are known to inhibit mammalian acetylcholinesterases thereby acting as nerve agents. Consequently, organophosphate-degrading enzymes are of considerable interest in light of their ability to detoxify such compounds. Here we report the three-dimensional structure of such an enzyme, namely, phosphotriesterase, as determined by single crystal X-ray diffraction analysis to 2.1-A resolution. Crystals employed in this investigation belonged to the space group P2(1)2(1)2 with unit cell dimensions of a = 80.3 A, b = 93.4 A, and c = 44.8 A and one molecule per asymmetric unit. The structure was solved by multiple isomorphous replacement with two heavy-atom derivatives and refined to a crystallographic R factor of 18.0%. As observed in various other enzymes, the overall fold of the molecule consists of an alpha/beta barrel with eight strands of parallel beta-pleated sheet. In addition, there are two antiparallel beta-strands at the N-terminus. The molecular model of phosphotriesterase presented here provides the initial structural framework necessary toward understanding the enzyme's broad substrate specificities and its catalytic mechanism.
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17. |
Ochi K,
( 1995 ) Comparative ribosomal protein sequence analyses of a phylogenetically defined genus, Pseudomonas, and its relatives. PMID : 7727274 : DOI : 10.1099/00207713-45-2-268 Abstract >>
I analyzed various families of ribosomal proteins obtained from selected species belonging to the genus Pseudomonas sensu stricto and allied organisms which were previously classified in the genus Pseudomonas. Partial amino acid sequencing of L30 preparations revealed that the strains which I examined could be divided into three clusters. The first cluster, which was assigned to the genus Pseudomonas sensu stricto, included Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas mendocina, and Pseudomonas fluorescens. The second cluster included Burkholderia pickettii and Burkholderia plantarii. The third cluster, which was a deeply branching cluster in the stem of gram-negative bacteria, included Brevundimonas diminuta and Brevundimonas vesicularis. Despite the different levels of conservation of the N-terminal sequences of ribosomal protein families (the highest level of similarity was 74% for L27 proteins and the lowest level of similarity was 42% for L30 proteins), similar phylogenetic trees were constructed by using data obtained from sequence analyses of various ribosomal protein families, including the S20, S21, L27, L29, L31, L32, and L33 protein families. Thus, I demonstrated the efficacy of ribosomal protein analysis in bacterial taxonomy.
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18. |
Lehmann M,
Tshisuaka B,
Fetzner S,
Lingens F,
( 1995 ) Molecular cloning of the isoquinoline 1-oxidoreductase genes from Pseudomonas diminuta 7, structural analysis of iorA and iorB, and sequence comparisons with other molybdenum-containing hydroxylases. PMID : 7782304 : DOI : 10.1074/jbc.270.24.14420 Abstract >>
The iorA and iorB genes from the isoquinoline-degrading bacterium Pseudomonas diminuta 7, encoding the heterodimeric molybdo-iron-sulfur-protein isoquinoline 1-oxidoreductase, were cloned and sequenced. The deduced amino acid sequences IorA and IorB showed homologies (i) to the small (gamma) and large (alpha) subunits of complex molybdenum-containing hydroxylases (alpha beta gamma/alpha 2 beta 2 gamma 2) possessing a pterin molybdenum cofactor with a monooxo-monosulfido-type molybdenum center, (ii) to the N- and C-terminal regions of aldehyde oxidoreductase from Desulfovibrio gigas, and (iii) to the N- and C-terminal domains of eucaryotic xanthine dehydrogenases, respectively. The closest similarity to IorB was shown by aldehyde dehydrogenase (Adh) from the acetic acid bacterium Acetobacter polyoxogenes. Five conserved domains of IorB were identified by multiple sequence alignments. Whereas IorB and Adh showed an identical sequential arrangement of these conserved domains, in all other molybdenum-containing hydroxylases the relative position of "domain A" differed. IorA contained eight conserved cysteine residues. The amino acid pattern harboring the four cysteine residues proposed to ligate the Fe/S I cluster was homologous to the consensus binding site of bacterial and chloroplast-type [2Fe-2S] ferredoxins, whereas the pattern including the four cysteines assumed to ligate the Fe/S II center showed no similarities to any described [2Fe-2S] binding motif. The N-terminal region of IorB comprised a putative signal peptide similar to typical leader peptides, indicating that isoquinoline 1-oxidoreductase is associated with the cell membrane.
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19. |
Benning MM,
Kuo JM,
Raushel FM,
Holden HM,
( 1995 ) Three-dimensional structure of the binuclear metal center of phosphotriesterase. PMID : 7794910 : DOI : 10.1021/bi00025a002 Abstract >>
Phosphotriesterase, as isolated from Pseudomonas diminuta, is capable of detoxifying widely used pesticides such as paraoxon and parathion and various mammalian acetylcholinesterase inhibitors. The enzyme requires a binuclear metal center for activity. Recently, the three-dimensional structure of the apoenzyme was solved (Benning et al., 1994) and shown to consist of an alpha/beta-barrel. Here we describe the three-dimensional structure of the holoenzyme, reconstituted with cadmium, as determined by X-ray crystallographic analysis to 2.0-A resolution. Crystals employed in the investigation belonged to the space group C2 with unit cell dimensions of a = 129.5 A, b = 91.4 A, c = 69.4 A, beta = 91.9 degrees, and two subunits in the asymmetric unit. There are significant differences in the three-dimensional architecture of the apo and holo forms of the enzyme such that their alpha-carbon positions superimpose with a root-mean-square deviation of 3.4 A. The binuclear metal center is located at the C-terminus of the beta-barrel with the cadmiums separated by 3.8 A. There are two bridging ligands to the metals: a water molecule (or possibly a hydroxide ion) and a carbamylated lysine residue (Lys 169). The more buried cadmium is surrounded by His 55, His 57, Lys 169, Asp 301, and the bridging water in a trigonal bipyramidal arrangement. The second metal is coordinated in a distorted octahedral geometry by His 201, His 230, Lys 169, the bridging water molecule, and two additional solvents.
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20. |
Kodani S,
Hemmi H,
Miyake Y,
Kaweewan I,
Nakagawa H,
( 2018 ) Heterologous production of a new lasso peptide brevunsin in Sphingomonas subterranea. PMID : 30191430 : DOI : 10.1007/s10295-018-2077-6 Abstract >>
A shuttle vector pHSG396Sp was constructed to perform gene expression using Sphingomonas subterranea as a host. A new lasso peptide biosynthetic gene cluster, derived from Brevundimonas diminuta, was amplified by PCR and integrated to afford a expression vector pHSG396Sp-12697L. The new lasso peptide brevunsin was successfully produced by S. subterranea, harboring the expression vector, with a high production yield (10.2 mg from 1 L culture). The chemical structure of brevunsin was established by NMR and MS/MS experiments. Based on the information obtained from the NOE experiment, the three-dimensional structure of brevunsin was determined, which indicated that brevunsin possessed a typical lasso structure. This expression vector system provides a new heterologous production method for unexplored lasso peptides that are encoded by bacterial genomes.
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21. |
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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