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Gao B,
Gupta RS,
( 2005 ) Conserved indels in protein sequences that are characteristic of the phylum Actinobacteria. PMID : 16280504 : DOI : 10.1099/ijs.0.63785-0 Abstract >>
Gram-positive bacteria with a high G+C content are currently recognized as a distinct phylum, Actinobacteria, on the basis of their branching in 16S rRNA trees. Except for an insert in the 23S rRNA, there are no unique biochemical or molecular characteristics known at present that can distinguish this group from all other bacteria. In this work, three conserved indels (i.e. inserts or deletions) are described in three widely distributed proteins that are distinctive characteristics of the Actinobacteria and are not found in any other groups of bacteria. The identified signatures are a 2 aa deletion in cytochrome-c oxidase subunit 1 (Cox1), a 4 aa insert in CTP synthetase and a 5 aa insert in glutamyl-tRNA synthetase (GluRS). Additionally, the actinobacterial specificity of the large insert in the 23S rRNA was also tested. Using primers designed for conserved regions flanking these signatures, fragments of most of these genes were amplified from 23 actinobacterial species, covering many different families and orders, for which no sequence information was previously available. All the 61 sequenced fragments, except two in GluRS, were found to contain the indicated signatures. The presence of these signatures in various species from 20 families within this phylum provides evidence that they are likely distinctive characteristics of the entire phylum, which were introduced in a common ancestor of this group. The absence of all four of these signatures in Symbiobacterium thermophilum suggests that this species, which is distantly related to other actinobacteria in 16S rRNA and CTP synthetase trees, may not be a part of the phylum Actinobacteria. The identified signatures provide novel molecular means for defining and circumscribing the phylum Actinobacteria. Functional studies on them should prove helpful in understanding novel biochemical and physiological characteristics of this group of bacteria.
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Ferraroni M,
Seifert J,
Travkin VM,
Thiel M,
Kaschabek S,
Scozzafava A,
Golovleva L,
Schlömann M,
Briganti F,
( 2005 ) Crystal structure of the hydroxyquinol 1,2-dioxygenase from Nocardioides simplex 3E, a key enzyme involved in polychlorinated aromatics biodegradation. PMID : 15772073 : DOI : 10.1074/jbc.M500666200 Abstract >>
Hydroxyquinol 1,2-dioxygenase (1,2-HQD) catalyzes the ring cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), a central intermediate in the degradation of aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants. We report here the primary sequence determination and the analysis of the crystal structure of the 1,2-HQD from Nocardioides simplex 3E solved at 1.75 A resolution using the multiple wavelength anomalous dispersion of the two catalytic irons (1 Fe/293 amino acids). The catalytic Fe(III) coordination polyhedron composed by the side chains of Tyr164, Tyr197, His221, and His223 resembles that of the other known intradiol-cleaving dioxygenases, but several of the tertiary structure features are notably different. One of the most distinctive characteristics of the present structure is the extensive openings and consequent exposure to solvent of the upper part of the catalytic cavity arranged to favor the binding of hydroxyquinols but not catechols. A co-crystallized benzoate-like molecule is also found bound to the metal center forming a distinctive hydrogen bond network as observed previously also in 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP. This is the first structure of an intradiol dioxygenase specialized in hydroxyquinol ring cleavage to be investigated in detail.
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Limburg J,
Mure M,
Klinman JP,
( 2005 ) Cloning and characterization of histamine dehydrogenase from Nocardioides simplex. PMID : 15752704 : DOI : 10.1016/j.abb.2004.11.024 Abstract >>
Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here, the hdh gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6+/-0.3 mol Fe and 3.7+/-0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histamine reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow substrate specificity, oxidizing only histamine (K(m)=31+/-11 microM, k(cat)/K(m)=2.1 (+/-0.4)x10(5)M(-1)s(-1)), agmatine (K(m)=37+/-6 microM, k(cat)/K(m)=6.0 (+/-0.6)x10(4)M(-1)s(-1)), and putrescine (K(m)=1280+/-240 microM, k(cat)/K(m)=1500+/-200 M(-1)s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K(m) (histamine) and the measurement of a substrate deuterium isotope effect, (D)(k(cat)/K(m) (histamine))=7.0+/-1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat))=1.3+/-0.2.
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4. |
Fujieda N,
Satoh A,
Tsuse N,
Kano K,
Ikeda T,
( 2004 ) 6-S-cysteinyl flavin mononucleotide-containing histamine dehydrogenase from Nocardioides simplex: molecular cloning, sequencing, overexpression, and characterization of redox centers of enzyme. PMID : 15311941 : DOI : 10.1021/bi049061q Abstract >>
Histamine dehydrogenase from Nocardioides simplex is a homodimeric enzyme and catalyzes oxidative deamination of histamine. The gene encoding this enzyme has been sequenced and cloned by polymerase chain reactions and overexpressed in Escherichia coli. The sequence of the complete open reading frame, 2073 bp coding for a protein of 690 amino acids, was determined on both strands. The amino acid sequence of histamine dehydrogenase is closely related to those of trimethylamine dehydrogenase and dimethylamine dehydrogenase containing an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-flavin mononucleotide, and one 4Fe-4S cluster as redox active cofactors in each subunit of the homodimer. The presence of the identical redox cofactors in histamine dehydrogenase has been confirmed by sequence alignment analysis, mass spectral analysis, UV-vis and EPR spectroscopy, and chemical analysis of iron and acid-labile sulfur. These results suggest that the structure of histamine dehydrogenase in the vicinity of the two redox centers is almost identical to that of trimethylamine dehydrogenase as a whole. The structure modeling study, however, demonstrated that a putative substrate-binding cavity in histamine dehydrogenase is quite distinct from that of trimethylamine dehydrogenase.
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5. |
Reed T,
Lushington GH,
Xia Y,
Hirakawa H,
Travis DM,
Mure M,
Scott EE,
Limburg J,
( 2010 ) Crystal structure of histamine dehydrogenase from Nocardioides simplex. PMID : 20538584 : DOI : 10.1074/jbc.M109.084301 PMC : PMC2919140 Abstract >>
Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-A resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S](2+)) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr(60), Trp(264), and Trp(355) provide the framework for the "aromatic bowl" that serves as a trimethylamine-binding site in TMADH is comprised of Gln(65), Trp(267), and Asp(358), respectively, in HADH. The surface Tyr(442) that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.
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( 1997 ) Characterization of an intradiol dioxygenase involved in the biodegradation of the chlorophenoxy herbicides 2,4-D and 2,4,5-T. PMID : 9141483 : DOI : 10.1016/s0014-5793(97)00297-4 Abstract >>
Hydroxyquinol 1,2-dioxygenase, an intradiol dioxygenase, which catalyzes the cleaving of the aromatic ring of hydroxyquinol, a key intermediate of 2,4-D and 2,4,5-T degradation, was purified from Nocardioides simplex 3E cells grown on 2,4-D as the sole carbon source. This enzyme exhibits a highly restricted substrate specificity and is able to cleave hydroxyquinol (K(m) for hydroxyquinol as a substrate was 1.2 microM, V(max) 55 U/mg, K(cat) 57 s-1 and K(cat)/K(m) 47.5 microM s-1), 6-chloro- and 5-chlorohydroxyquinol. Different substituted catechols and hydroquinones are not substrates for this enzyme. This enzyme appears to be a dimer with two identical 37-kDa subunits. Protein and iron analyses indicate an iron stoichiometry of 1 iron/65 kDa homodimer, alpha2 Fe. Both the electronic absorption spectrum which shows a broad absorption band with a maximum at 450 nm and the electron paramagnetic resonance spectra are consistent with a high-spin iron(III) ion in a rhombic environment typical of the active site of intradiol cleaving enzymes.
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7. |
Molnár I,
Choi KP,
Yamashita M,
Murooka Y,
( 1995 ) Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-delta 1-dehydrogenase, 3-ketosteroid-delta 5-isomerase and a hypothetical regulatory protein. PMID : 7596291 : DOI : 10.1111/j.1365-2958.1995.tb02359.x Abstract >>
The Arthrobacter simplex gene coding for 3-ketosteroid-delta 1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned in Streptomyces lividans. Nucleotide sequence analysis revealed that the gene for 3-ketosteroid-delta 1-dehydrogenase (ksdD) is clustered with at least two more genes possibly involved in steroid metabolism. Upstream of ksdD, we found a gene, ksdR, encoding a hypothetical regulatory protein that shows homologies to KdgR, the negative regulator of pectin biodegradation in Erwinia, and GyIR, the activator for glycerol metabolism in Steptomyces. A helix-turn-helix DNA-binding domain can be predicted at similar positions near the N-terminal of KsdR, KdgR and GyIR. ksdl adjoining downstream to ksdD codes for a protein that has strong similarities to 3-ketosteroid-delta 5-isomerases. The highly conserved Tyr and Asp residues are present in the active-centre motif of the enzyme. The translated ksdD gene product was found to be similar to the 3-ketosteroid-delta 1-dehydrogenase of Pseudomonas testosteroni and to the fumarate reductase of Shewanella putrefaciens. A region highly conserved between the two steroid dehydrogenases can be aligned to the active-centre motif of the fumarate reductase. S. lividans strains carrying the ksdD gene overexpressed 3-ketosteroid-delta 1-dehydrogenase. The expression of 3-ketosteroid-delta 5-isomerase, however, was barely detectable in recombinant S. lividans strains carrying the ksdl gene, or in the parental Arthrobacter strain.
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