Degradation of 2-nitrobenzoate by Burkholderia terrae strain KU-15

Bacterial strain KU-15, identified as a Burkholderia terrae by 16S rRNA gene sequence analysis, was one of 11 new isolates that grew on 2-nitrobenzoate as sole source of carbon and nitrogen. Strain KU-15 was also found to grow on anthranilate, 4-nitrobenzoate, and 4-aminobenzoate. Whole cells of strain KU-15 were found to accumulate ammonia in the medium, indicating that the degradation of 2-nitrobenzoate proceeds through a reductive route. Metabolite analyses by high-performance liquid chromatography indicated that 3-hydroxyanthranilate, anthranilate, and catechol are intermediates of 2-nitrobenzoate metabolism in strain KU-15. Enzyme studies suggested that 2-nitrobenzoate degradation occurs via the formation of 2-hydroxylaminobenzoate and that the pathway branches at this point to form two different aromatic intermediates: anthranilate and 3-hydroxyanthranilate. PCR amplifications and DNA sequencing revealed DNA fragments encoding a polypeptide homologous to 2-amino-3-carboxymuconate 6-semialdehyde decarboxylase and anthranilate 1,2-dioxygenase.     

A novel coupled enzyme assay reveals an enzyme responsible for the deamination of a chemically unstable intermediate in the metabolic pathway of 4-amino-3-hydroxybenzoic acid in Bordetella sp. strain 10d.

2-amino-5-carboxymuconic 6-semialdehyde is an unstable intermediate in the meta-cleavage pathway of 4-amino-3-hydroxybenzoic acid in Bordetella sp. strain 10d. In vitro, this compound is nonenzymatically converted to 2,5-pyridinedicarboxylic acid. Crude extracts of strain 10d grown on 4-amino-3-hydroxybenzoic acid converted 2-amino-5-carboxymuconic 6-semialdehyde formed from 4-amino-3-hydroxybenzoic acid by the first enzyme in the pathway, 4-amino-3-hydroxybenzoate 2,3-dioxygenase, to a yellow compound (epsilonmax = 375 nm). The enzyme in the crude extract carrying out the next step was purified to homogeneity. The yellow compound formed from 4-amino-3-hydroxybenzoic acid by this purified enzyme and purified 4-amino-3-hydroxybenzoate 2,3-dioxygenase in a coupled assay was identified as 2-hydroxymuconic 6-semialdehyde by GC-MS analysis. A mechanism for the formation of 2-hydroxymuconic 6-semialdehyde via enzymatic deamination and nonenzymatic decarboxylation is proposed based on results of spectrophotometric analyses. The purified enzyme, designated 2-amino-5-carboxymuconic 6-semialdehyde deaminase, is a new type of deaminase that differs from the 2-aminomuconate deaminases reported previously in that it primarily and specifically attacks 2-amino-5-carboxymuconic 6-semialdehyde. The deamination step in the proposed pathway differs from that in the pathways for 2-aminophenol and its derivatives.     

Metabolism of 4-amino-3-hydroxybenzoic acid by Bordetella sp. strain 10d: A different modified meta-cleavage pathway for 2-aminophenols

Bordetella sp. strain 10d metabolizes 4-amino-3-hydroxybenzoic acid via 2-hydroxymuconic 6-semialdehyde. Cell extracts from 4-amino-3-hydroxybenzoate-grown cells showed high NAD(+)-dependent 2-hydroxymuconic 6-semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase activities, but no 2-hydroxymuconic 6-semialdehyde hydrolase activity. These enzymes involved in 4-amino-3-hydroxybenzoate metabolism were purified and characterized. When 2-hydroxymuconic 6-semialdehyde was used as substrate in a reaction mixture containing NAD(+) and cell extracts from 4-amino-3-hydroxybenzoate-grown cells, 4-oxalocrotonic acid, 2-oxopent-4-enoic acid, and 4-hydroxy-2-oxovaleric acid were identified as intermediates, and pyruvic acid was identified as the final product. A complete pathway for the metabolism of 4-amino-3-hydroxybenzoic acid in strain 10d is proposed. Strain 10d metabolized 2-hydroxymuconic 6-semialdehyde derived from 4-amino-3-hydroxybenzoic acid via a dehydrogenative route, not via a hydrolytic route. This proposed metabolic pathway differs considerably from the modified meta-cleavage pathway of 2-aminophenol and those previously reported for methyl- and chloro-derivatives.     

Branching of o-nitrobenzoate degradation pathway in Arthrobacter protophormiae RKJ100: identification of new intermediates

We have earlier reported a novel reductive pathway for o-nitrobenzoate (ONB) degradation (at 0.5 mM) in Arthrobacter protophormiae RKJ100, which proceeds via the formation of o-hydroxylaminobenzoate (HABA) and anthranilate (AA). During growth of this organism at 40 times higher concentration (20 mM) of ONB, 3-hydroxyanthranilate (HAA) was identified as an intermediate by thin layer chromatography, gas chromatography and high performance liquid chromatography studies. Crude cell extracts of ONB-grown cells showed HAA 3,4-dioxygenase activity suggesting HAA as a terminal aromatic intermediate of the catabolic energy-yielding pathway as shown before in Pseudomonas fluorescens strain KU-7. HAA is further cleaved to 2-amino-3-carboxymuconic-6-semialdehyde by the action of HAA 3,4-dioxygenase. In this report we propose that ONB degradation occurs via the formation of HABA and the pathway branches at this point to form the two different aromatic intermediates AA and HAA by the action of a reductase and a mutase, respectively.     

A novel meta-cleavage dioxygenase that cleaves a carboxyl-group-substituted 2-aminophenol. Purification and characterization of 4-amino-3-hydroxybenzoate 2,3-dioxygenase from Bordetella sp. strain 10d.

A bacterial strain that grew on 4-amino-3-hydroxybenzoic acid was isolated from farm soil. The isolate, strain 10d, was identified as a species of Bordetella. Cell extracts of Bordetella sp. strain 10d grown on 4-amino-3-hydroxybenzoic acid contained an enzyme that cleaved this substrate. The enzyme was purified to homogeneity with a 110-fold increase in specific activity. The purified enzyme was characterized as a meta-cleavage dioxygenase that catalyzed the ring fission between C2 and C3 of 4-amino-3-hydroxybenzoic acid, with the consumption of 1 mol of O2 per mol of substrate. The enzyme was therefore designated as 4-amino-3-hydroxybenzoate 2,3-dioxygenase. The molecular mass of the native enzyme was 40 kDa based on gel filtration; the enzyme is composed of two identical 21-kDa subunits according to SDS/PAGE. The enzyme showed a high dioxygenase activity only for 4-amino-3-hydroxybenzoic acid. The Km and Vmax values for this substrate were 35 micro m and 12 micro mol.min-1.(mg protein)-1, respectively. Of the 2-aminophenols tested, only 4-aminoresorcinol and 6-amino-m-cresol inhibited the enzyme. The enzyme reported here differs from previously reported extradiol dioxygenases, including 2-aminophenol 1,6-dioxygenase, in molecular mass, subunit structure and catalytic properties.     

Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from Pseudomonas pseudoalcaligenes JS45

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD⁺. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.     

Gene cloning and characterization of a deaminase from the 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d

The 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d produces a deaminase that catalyzes the deamination of 2-amino-5-carboxymuconic 6-semialdehyde. A gene encoding the deaminase, ahdB , was cloned and expressed in Escherichia coli; ahdB is located downstream from the previously reported genes encoding 4-amino-3-hydroxybenzoate 2,3-dioxygenase ( ahdA ) and a LysR-type regulator. The deduced amino acid sequence of ahdB shows 30–33% identity to those of previously reported 2-aminomuconate deaminases. We identified a region (RAGDFLXVSG) conserved in AhdB and three other deaminases. The recombinant enzyme AhdB was purified to homogeneity. After a coupled enzyme assay with purified AhdA, AhdB, and the substrate 4-amino-3-hydroxybenzoate, the final product, formed by the action of AhdA, AhdB, and by nonenzymatic decarboxylation, was identified by HPLC, MS, and ¹H-nuclear magnetic resonance analyses as 2-hydroxymuconic 6-semialdehyde.     

Characterization of a pseudomonad 2-nitrobenzoate nitroreductase and its catabolic pathway-associated 2-hydroxylaminobenzoate mutase and a chemoreceptor involved in 2-nitrobenzoate chemotaxis

Pseudomonas fluorescens strain KU-7 is a prototype microorganism that metabolizes 2-nitrobenzoate (2-NBA) via the formation of 3-hydroxyanthranilate (3-HAA), a known antioxidant and reductant. The initial two steps leading to the sequential formation of 2-hydroxy/aminobenzoate and 3-HAA are catalyzed by a NADPH-dependent 2-NBA nitroreductase (NbaA) and 2-hydroxylaminobenzoate mutase (NbaB), respectively. The 216-amino-acid protein NbaA is 78% identical to a plasmid-encoded hypothetical conserved protein of Polaromonas strain JS666; structurally, it belongs to the homodimeric NADH:flavin mononucleotide (FMN) oxidoreductase-like fold family. Structural modeling of complexes with the flavin, coenzyme, and substrate suggested specific residues contributing to the NbaA catalytic activity, assuming a ping-pong reaction mechanism. Mutational analysis supports the roles of Asn40, Asp76, and Glu113, which are predicted to form the binding site for a divalent metal ion implicated in FMN binding, and a role in NADPH binding for the 10-residue insertion in the beta5-alpha2 loop. The 181-amino-acid sequence of NbaB is 35% identical to the 4-hydroxylaminobenzoate lyases (PnbBs) of various 4-nitrobenzoate-assimilating bacteria, e.g., Pseudomonas putida strain TW3. Coexpression of nbaB with nbaA in Escherichia coli produced a small amount of 3-HAA from 2-NBA, supporting the functionality of the nbaB gene. We also showed by gene knockout and chemotaxis assays that nbaY, a chemoreceptor NahY homolog located downstream of the nbaA gene, is responsible for strain KU-7 being attracted to 2-NBA. NbaY is the first chemoreceptor in nitroaromatic metabolism to be identified, and this study completes the gene elucidation of 2-NBA metabolism that is localized within a 24-kb chromosomal locus of strain KU-7.     

Identification of a Toxic Mechanism of the Plasticizers,Phtahlic Acid Esters,which are Putative Endocrine Disrupters: Time-dependent Increase in Quinolinic Acid and Its Metabolites in…

We have reported that the administration of di(2-ethylhexyl)phthalate (DEHP) increased the formations of quinolinic acid (QA) and its lower metabolites on the tryptophan-niacin pathway. To discover the mechanism involved in disruption of the tryptophan-niacin pathway by DEHP, we assessed the daily urinary excretion of QA and its lower metabolites, and enzyme activities on the tryptophan-niacin pathway. Rats were fed with a niacin-free, 20% casein diet or the same diet supplemented with 0.1% DEHP or 0.043% phthalic acid and 0.067% 2-ethylhexanol added for 21 days. Feeding of DEHP increased the urinary excretions of QA and its lower metabolites in a time-dependent manner, and the increase of these excretions reached a peak at 11 days, but feeding of phthalic acid and 2-ethylhexanol had no effect. Feeding of DEHP, however, did not affect any enzyme activity including α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), affecting the formation of QA, on the tryptophan-niacin pathway.     

Complete nucleotide sequence and functional analysis of the genes for 2-aminophenol metabolism from Pseudomonas sp. AP-3

A 13.9-kb region, which contained the 2-aminophenol 1,6-dioxygenase genes (amnBA) reported before, was cloned from the 2-aminophenol-assimilating bacterium Pseudomonas sp. AP-3. The complete nucleotide sequence of this region was determined and six genes were found downstream of amnBA. The eight genes together were designated amnBACFEDHG. Each gene was similar to the corresponding gene operating in the meta-cleavage pathway, except for amnB, amnA, and amnD. The four 2-aminophenol-metabolizing enzymes, 2-aminomuconic 6-semialdehyde dehydrogenase, 2-aminomuconate deaminase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase, were purified and characterized. NH2-terminal amino acid sequences of each purified enzyme agreed with those deduced from amnC, amnF, amnE, and amnD, respectively. These genes were therefore assigned as the genes encoding these respective proteins. The tight clustering of the amn genes, which were all transcribed in the same direction, raised the possibility that these genes formed a single operon. The organization of the amn genes was entirely different from that of the atd, dmp, and xyl genes reported in the meta-cleavage pathway, although these latter genes clustered similarly.     

Effects of dietary di(2-ethylhexyl)phthalate, a putative endocrine disrupter, on enzyme activities involved in the metabolism of tryptophan to niacin in rats

We have reported that the conversion ratio of tryptophan to niacin increased with increasing dietary concentration of di(2-ethylhexyl)phthalate (DEHP); the conversion ratio was about 2.0% in the control rat, which increased by about 30% in the rat fed with 3.0% DEHP diet. In this study, we investigated whether this abnormal increase in the conversion ratio by DEHP occurred through the alteration of the enzyme activities involved in the metabolism of tryptophan to niacin. Rats were fed with a diet containing 0%, 0.1%, 0.5%, or 1.0% DEHP for 21 days. The nine kinds of enzyme activities involved in the biosynthesis and catabolism in the liver and kidney were measured. Based on previous findings that the formation of quinolinic acid and its' metabolites significantly increased with DEHP administration, we proposed that the activity of 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase would be inhibited by DEHP intake. However, we found that the activities in the liver and kidney did not decrease in the rat fed with DEHP-containing diet. We discuss the discrepancy between the metabolite results and the enzyme activities.     

Glutamyl-tRNA reductase of Chlorobium vibrioforme is a dissociable homodimer that contains one tightly bound heme per subunit

delta-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His(6) tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His(6)-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNA(Glu) and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of beta-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.     

Structural studies on 3-hydroxyanthranilate-3,4-dioxygenase: the catalytic mechanism of a complex oxidation involved in NAD biosynthesis

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) catalyzes the oxidative ring opening of 3-hydroxyanthranilate in the final enzymatic step of the biosynthetic pathway from tryptophan to quinolinate, the universal de novo precursor to the pyridine ring of nicotinamide adenine dinucleotide. The enzyme requires Fe2+ as a cofactor and is inactivated by 4-chloro-3-hydroxyanthranilate. HAD from Ralstonia metallidurans was crystallized, and the structure was determined at 1.9 A resolution. The structures of HAD complexed with the inhibitor 4-chloro-3-hydroxyanthranilic acid and either molecular oxygen or nitric oxide were determined at 2.0 A resolution, and the structure of HAD complexed with 3-hydroxyanthranilate was determined at 3.2 A resolution. HAD is a homodimer with a subunit topology that is characteristic of the cupin barrel fold. Each monomer contains two iron binding sites. The catalytic iron is buried deep inside the beta-barrel with His51, Glu57, and His95 serving as ligands. The other iron site forms an FeS4 center close to the solvent surface in which the sulfur atoms are provided by Cys125, Cys128, Cys162, and Cys165. The two iron sites are separated by 24 A. On the basis of the crystal structures of HAD, mutagenesis studies were carried out in order to elucidate the enzyme mechanism. In addition, a new mechanism for the enzyme inactivation by 4-chloro-3-hydroxyanthranilate is proposed.     

Metabolism of 2-aminophenol by Pseudomonas sp. AP-3: modified meta-cleavage pathway

A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2,4-pentadienoic acid is the last metabolite containing an amino group. We, therefore, propose a modified meta-cleavage pathway for the 2-aminophenol metabolism. Received: 27 November 1997 / Accepted: 14 May 1998     

Reactions involved in the lower pathway for degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1

In spite of the variety of initial reactions, the aerobic biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the degradation of nitroaromatic compounds revealed that some nitroaromatic compounds are initially converted to 2-aminophenol rather than dihydroxy intermediates by a number of microorganisms. The complete pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 has been elucidated previously. The pathway is parallel to the catechol extradiol ring cleavage pathway, except that 2-aminophenol is the ring cleavage substrate. Here we report the elucidation of the pathway of 2-amino-4-methylphenol (6-amino-m-cresol) metabolism during the degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1 and the comparison of the substrate specificities of the relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate that the 2-aminophenol ring cleavage pathway in strain JS45 is not unique but is representative of the pathways of metabolism of other o-aminophenolic compounds.     

Reactions Involved in the Lower Pathway for Degradation of 4-Nitrotoluene by Mycobacterium Strain HL 4-NT-1

In spite of the variety of initial reactions, the aerobic biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the degradation of nitroaromatic compounds revealed that some nitroaromatic compounds are initially converted to 2-aminophenol rather than dihydroxy intermediates by a number of microorganisms. The complete pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 has been elucidated previously. The pathway is parallel to the catechol extradiol ring cleavage pathway, except that 2-aminophenol is the ring cleavage substrate. Here we report the elucidation of the pathway of 2-amino-4-methylphenol (6-amino-m-cresol) metabolism during the degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1 and the comparison of the substrate specificities of the relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate that the 2-aminophenol ring cleavage pathway in strain JS45 is not unique but is representative of the pathways of metabolism of other o-aminophenolic compounds.     

The mechanism of inactivation of 3-hydroxyanthranilate-3,4-dioxygenase by 4-chloro-3-hydroxyanthranilate

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) is a non-heme Fe(II) dependent enzyme that catalyzes the oxidative ring-opening of 3-hydroxyanthranilate to 2-amino-3-carboxymuconic semialdehyde. The enzymatic product subsequently cyclizes to quinolinate, an intermediate in the biosynthesis of nicotinamide adenine dinucleotide. Quinolinate has also been implicated in important neurological disorders. Here, we describe the mechanism by which 4-chloro-3-hydroxyanthranilate inhibits the HAD catalyzed reaction. Using overexpressed and purified bacterial HAD, we demonstrate that 4-chloro-3-hydroxyanthranilate functions as a mechanism-based inactivating agent. The inactivation results in the consumption of 2 +/- 0.8 equiv of oxygen and the production of superoxide. EPR analysis of the inactivation reaction demonstrated that the inhibitor stimulated the oxidation of the active site Fe(II) to the catalytically inactive Fe(III) oxidation state. The inactivated enzyme can be reactivated by treatment with DTT and Fe(II). High resolution ESI-FTMS analysis of the inactivated enzyme demonstrated that the inhibitor did not form an adduct with the enzyme and that four conserved cysteines were oxidized to two disulfides (Cys125-Cys128 and Cys162-Cys165) during the inactivation reaction. These results are consistent with a mechanism in which the enzyme, complexed to the inhibitor and O2, generates superoxide which subsequently dissociates, leaving the inhibitor and the oxidized iron center at the active site.     

2-aminophenol 1,6-dioxygenase: a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS45.

Most bacterial pathways for the degradation of aromatic compounds involve introduction of two hydroxyl groups either ortho or para to each other. Ring fission then occurs at the bond adjacent to one of the hydroxyl groups. In contrast, 2-aminophenol is cleaved to 2-aminomuconic acid semialdehyde in the nitrobenzene-degrading strain Pseudomonas pseudoalcaligenes JS45. To examine the relationship between this enzyme and other dioxygenases, 2-aminophenol 1,6-dioxygenase has been purified by ethanol precipitation, gel filtration, and ion exchange chromatography. The molecular mass determined by gel filtration was 140,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two subunits of 35,000 and 39,000 Da, which suggested an alpha2beta2 subunit structure. Studies with inhibitors indicated that ferrous iron was the sole cofactor. The Km values for 2-aminophenol and oxygen were 4.2 and 710 microM, respectively. The enzyme catalyzed the oxidation of catechol, 6-amino-m-cresol, 2-amino-m-cresol, and 2-amino-4-chlorophenol. 3-Hydroxyanthranilate, protocatechuate, gentisate, and 3- and 4-methylcatechol were not substrates. The substrate range and the subunit structure are unique among those of the known ring cleavage dioxygenases.     

Thermophilic, reversible gamma-resorcylate decarboxylase from Rhizobium sp. strain MTP-10005: purification, molecular characterization, and expression

We found the occurrence of thermophilic reversible γ-resorcylate decarboxylase (γ-RDC) in the cell extract of a bacterium isolated from natural water, Rhizobium sp. strain MTP-10005, and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be about 151 kDa by gel filtration, and that of the subunit was 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; in other words, the enzyme was a homotetramer. The enzyme was induced specifically by the addition of γ-resorcylate to the medium. The enzyme required no coenzyme and did not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. It was relatively thermostable to heat treatment, and its half-life at 50°C was estimated to be 122 min; furthermore, it catalyzed the reverse carboxylation of resorcinol. The values of k_cat/K_m (mΜ⁻¹·s⁻¹) for γ-resorcylate and resorcinol at 30°C and pH 7 were 13.4 and 0.098, respectively. The enzyme contains 327 amino acid residues, and sequence identities were found with those of hypothetical protein AGR C 4595p from Agrobacterium tumefaciens strain C58 (96% identity), 5-carboxyvanillate decarboxylase from Sphingomonas paucimobilis (32%), and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylases from Bacillus cereus ATCC 10987 (26%), Rattus norvegicus (26%), and Homo sapiens (25%). The genes (graA [1,230 bp], graB [888 bp], and graC [1,056 bp]) that are homologous to those in the resorcinol pathway also exist upstream and downstream of the γ-RDC gene. Judging from these results, the resorcinol pathway also exists in Rhizobium sp. strain MTP-10005, and γ-RDC probably catalyzes a reaction just before the hydroxylase in it does.     

The tetrameric assembly of 2-aminomuconic 6-semialdehyde dehydrogenase is a functional requirement of cofactor NAD+ binding

The bacterium Pseudomonas sp. AP-3 is able to use the environmental pollutant 2-aminophenol as its sole source of carbon, nitrogen, and energy. Eight genes (amnA, B, C, D, E, F, G, and H) encoding 2-aminophenol metabolizing enzymes are clustered into a single operon. 2-Aminomuconic 6-semialdehyde dehydrogenase (AmnC), a member of the aldehyde dehydrogenase (ALDH) superfamily, is responsible for oxidizing 2-aminomuconic 6-semialdehyde to 2-aminomuconate. In contrast to many other members of the ALDH superfamily, the structural basis of the catalytic activity of AmnC remains elusive. Here, we present the crystal structure of AmnC, which displays a homotetrameric quaternary assembly that is directly involved in its enzymatic activity. The tetrameric state of AmnC in solution was also presented using small-angle X-ray scattering. The tetramerization of AmnC is mediated by the assembly of a protruding hydrophobic beta-strand motif and residues V121 and S123 located in the NAD⁺-binding domain of each subunit. Dimeric mutants of AmnC dramatically lose NAD⁺ binding affinity and failed to oxidize the substrate analogue 2-hydroxymuconate-6-semialdehyde to α-hydroxymuconic acid, indicating that tetrameric assembly of AmnC is functional requirement.