Regioselective hydroxylation of quinolinic acid,lutidinic acid and isocinchomeronic acid by resting cells of pyridine dicarboxylic acid-degrading microorganisms

Microorganisms aerobically degrading quinolinic acid, lutidinic acid or isocinchomeronic acid were isolated and the microbial regioselective hydroxylation of these pyridine dicarboxylic acids was studied. Alcaligenes sp. UK21 cells converted quinolinic acid into 6-hydroxypicolinic acid, suggesting the involvement of two enzyme reactions catalyzing hydroxylation at position C6 and decarboxylation at position C3 of quinolinic acid. Resting cells of Alcaligenes sp. UK21 accumulated 94.9 mM 6-hydroxypicolinic acid (13.2 g l(-1)), with a 96% molar conversion yield by 48 h incubation. Rhizobium sp. LA17 and Hydrogenophaga sp. IMA01 catalyzed the regioselective hydroxylation of lutidinic acid and isocinchomeronic acid into 6-hydroxylutidinic acid and 6-hydroxyisocinchomeronic acid, respectively. 6-Hydroxylutidinic acid accumulated up to 95.4 mM (17.5 g l(-1)) by 24 h incubation in the resting cells reaction, using Rhizobium sp. LA17, with a 99% molar conversion yield. Resting cells of Hydrogenophaga sp. IMA01 produced 88.7 mM 6-hydroxyisocinchomeronic acid (16.2 g l(-1)) by 24 h incubation, with a 81% molar conversion yield.     

Purification and characterization of 2,6-dihydroxybenzoate decarboxylase reversibly catalyzing nonoxidative decarboxylation

A nonoxidative decarboxylase, 2,6-dihydroxybenzoate decarboxylase, was found in Agrobacterium tumefaciens IAM12048. The enzyme activity was induced specifically by 2,6-dihydroxybenzoate. The purified enzyme was a homotetramer of identical 38 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 2,6-dihydroxybenzoate and 2,3-dihydroxybenzoate without requiring any cofactors. In the presence of KHCO₃, the enzyme also catalyzed the regioselective carboxylation of 1,3-dihydroxybenzene into 2,6-dihydroxybenzoate at a molar conversion ratio of 30%.     

Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240

We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K _m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K _m value was 6.80 mM. In the presence of 3 M KHCO₃ and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K _m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.     

Reversible and nonoxidative gamma-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and beta-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE.     

Desilicification from the High Silica Containing Kraft Black Liquor by the Improved Carbon Dioxide Method

Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO₂ production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system.

The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.     

The effect of pyrazines on the metabolism of tryptophan and nicotinamide adenine dinucleotide in the rat Evidence of the formation of a potent inhibitor of aminocarboxy-muconate-semialdehyde decarboxylase from pyrazinamide

The intraperitoneal or oral administration of pyrazinamide and pyrazinoic acid (pyrazine 2-carboxylic acid) resulted in a marked increase of the NAD content in rat liver. The injections of pyrazine and pyrazine 2,3-dicarboxylic acid exhibited no significant effect on the hepatic NAD content. The boiled extract obtained from liver and kidney of rat injected with either pyrazinamide or pyrazinoic acid exhibited a potent inhibitory effect on the aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) activity in either liver or kidney, although pyrazinamide or pyrazinoic acid per se did not inhibit the enzyme activity. The unknown inhibitor of aminocarboxymuconate-semialdehyde decarboxylase was dialysable and heat-stable, and mostly excreted in urine by 6 and 12 h after injection of pyrazinoic acid and pyrazinamide, respectively. Pyrazine 2,3-dicarboxylic acid, pyrazine, nicotinamide, nicotinic acid, tryptophan, anthranilic acid, 5-hydroxyanthranilic acid and quinolinic acid exhibited no significant effect on the aminocarboxymuconate-semialdehyde decarboxylase activity in liver and kidney at the concentration of 1 mM in the reaction mixture. The expired ¹⁴CO₂ from l-[benzen ring-U-¹⁴C]tryptophan was markedly decreased by the pyrazinamide injection, while the urinary excretion of ¹⁴C-labeled metabolites from l-tryptophan, mainly quinolinic acid, was markedly increased. These results suggest that the glutarate pathway of l-tryptophan was strongly inhibited by the inhibitor produced after the administration of pyrazinoic acid and pyrazinamide. Pyrazinamide but not pyrazinoic acid also exhibited a significant inhibition of the nuclear enzyme poly(ADP-ribose) synthetase in rat liver.     

The effect of pyrazines on the metabolism of tryptophan and nicotinamide adenine dinucleotide in the rat. Evidence of the formation of a potent inhibitor of aminocarboxy-muconate-semialdehyde decarboxylase from pyrazinamide

The intraperitoneal or oral administration of pyrazinamide and pyrazinoic acid (pyrazine 2-carboxylic acid) resulted in a marked increase of the NAD content in rat liver. The injections of pyrazine and pyrazine 2,3-dicarboxylic acid exhibited no significant effect on the hepatic NAD content. The boiled extract obtained from liver and kidney of rat injected with either pyrazinamide or pyrazinoic acid exhibited a potent inhibitory effect on the aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) activity in either lier or kidney, although pyrazinamide or pyrazinoic acid per se did not inhibit the enzyme activity. The unknown inhibitor of aminocarboxymuconate-semialdehyde decarboxylase was dialysable and heat-stable, and mostly excreted in urine by 6 and 12 h after injected of pyrazinoic acid and pyrazinamide, respectively. Pyrazine 2,3-dicarboxylic acid, pyrazine, nicotinamide, nicotinic acid, tryptophan, anthranilic acid, 5-hydroxyanthranilic acid and quinolinic acid exhibited no significant effect on the aminocarboxymuconate-semialdehyde decarboxylase activity in liver and kidney at the concentration of 1 mM in the reaction mixture. The expired 14CO2 from L-[benzen ring-U-14C]tryptophan was markedly decreased by the pyrazinamide injection, while the urinary excretion of 14C-labeled metabolites from L-tryptophan, mainly quinolinic acid, was markedly increased. These results suggest that the glutarate pathway of L-tryptophan was strongly inhibited by the inhibitor produced after the administration of pyrazinoic acid and pyrazinamide. Pyrazinamide but not pyrazinoic acid also exhibited a significant inhibition of the nuclear enzyme poly(ADP-ribose) synthetase in rat liver.     

Pusillimonas sp. 5HP degrading 5-hydroxypicolinic acid

A bacterial strain 5HP capable of degrading and utilizing 5-hydroxypicolinic acid as the sole source of carbon and energy was isolated from soil. In addition, the isolate 5HP could also utilize 3-hydroxypyridine and 3-cyanopyridine as well as nicotinic, benzoic and p-hydroxybenzoic acids for growth in the basic salt media. On the basis of 16S rRNA gene sequence analysis, the isolate 5HP was shown to belong to the genus Pusillimonas. Both the bioconversion analysis using resting cells and the enzymatic assay showed that the degradation of 5-hydroxypicolinic acid, 3-hydroxypyridine and nicotinic acid was inducible and proceeded via formation of the same metabolite, 2,5-dihydroxypyridine. The activity of a novel enzyme, 5-hydroxypicolinate 2-monooxygenase, was detected in the cell-free extracts prepared from 5-hydroxypicolinate-grown cells. The enzyme was partially purified and was shown to catalyze the oxidative decarboxylation of 5-hydroxypicolinate to 2,5-dihydroxypyridine. The activity of 5-hydroxypicolinate 2-monooxygenase was dependent on O₂, NADH and FAD.     

A molybdenum-containing dehydrogenase catalyzing an unusual 2-hydroxylation of nicotinic acid

An enzyme of Ralstonia/ Burkholderia strain DSM 6920 catalyzing the initial hydroxylation of 6-methylnicotinic acid at position 2 was purified to apparent homogeneity. It also catalyzed the unusual conversion of nicotinic acid to 2-hydroxynicotinic acid and was therefore designated as nicotinic acid dehydrogenase (NDH). Native NDH had a molecular mass of 280 kDa and was composed of subunits of 75, 30 and 16 kDa. It contained molybdenum, iron, acid-labile sulfur and FAD in a ratio of 1.6:7.3:8.0:0.6 mol(-1) of native enzyme. The molybdenum cofactor was characterized as molybdopterin cytosine dinucleotide. Zinc was identified as an additional metal ion in a molar ratio of 1.8 mol mol(-1) of native enzyme. Purified NDH exhibited a maximal specific activity of 22.6 micromol nitro blue tetrazoliumchloride reduced min(-1) mg(-1) of protein, using nicotinic acid as electron donor. The apparent K(m) value for nicotinic acid was determined to be 154 microM. Pyridine-3,5-dicarboxylic acid and quinoline-3-carboxylic acid were further substrates, but exhibited significantly different activity pH optima. Several artificial electron acceptors were reduced by NDH, but no activity was detected with NAD or O(2). NDH was inactivated upon incubation with cyanide, but no loss of activity was obtained in the presence of arsenite.     

Enzyme-catalysed non-oxidative decarboxylation of aromatic acids: I. Purification and spectroscopic properties of 2,3 dihydroxybenzoic acid decarboxylase from Aspergillus niger

In order to understand the molecular mechanism of non-oxidative decarboxylation of aromatic acids observed in microbial systems, 2,3 dihydroxybenzoic acid (DHBA) decarboxylase from Aspergillus niger was purified to homogeneity by affinity chromatography. The enzyme (Mr 120 kDa) had four identical subunits (28 kDa each) and was specific for DHBA. It had a pH optimum of 5.2 and Km was 0.34 mM. The decarboxylation did not require any cofactors, nor did the enzyme had any pyruvoyl group at the active site. The carboxyl group and hydroxyl group in the ortho-position were required for activity. The preliminary spectroscopic properties of the enzyme are also reported.     

Purification and properties of the formate dehydrogenase and characterization of the<Emphasis Type="Italic"> fdhA</Emphasis> gene of<Emphasis Type="Italic"> Sulfurospirillum multivorans</Emphasis>

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene (fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe₄/S₄ cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.Abbreviations Fdh Formate dehydrogenase - PCE Tetrachloroethene     

Purification and properties of oxaloacetate decarboxylase fromCorynebacterium glutamicum

Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated fromCorynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 ± 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an ₄ subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO₂, but no other -ketoacids were used as substrate. The cation Mn²⁺ was required for full activity, but could be substituted by Mg²⁺, Co²⁺, Ni²⁺ and Ca²⁺. Monovalent ions like Na⁺, K⁺ or NH ₄ ⁺ were not required for activity. The enzyme was inhibited by Cu²⁺, Zn²⁺, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a K _m for OAA of 2.1 mM and a K _m of 1.2 mM for Mn²⁺. The V _max was 158 µmol of OAA converted per min per mg of protein, which corresponds to an apparent k _cat of 311 s^–1.Abbreviations OAA oxaloacetate - LDH lactate dehydrogenase     

A high affinity pyruvate decarboxylase is present in cottonwood leaf veins and petioles: A second source of leaf acetaldehyde emission?

Considerable evidence indicates that acetaldehyde is released from the leaves of a variety of plants. The conventional explanation for this is that ethanol formed in the roots is transported to the leaves where it is converted to acetaldehyde by the alcohol dehydrogenase (ADH) found in the leaves. It is possible that acetaldehyde could also be formed in leaves by action of pyruvate decarboxylase (PDC), an enzyme with an uncertain metabolic role, which has been detected, but not characterized, in cottonwood leaves. We have found that leaf PDC is present in leaf veins and petioles, as well as in non-vein tissues. Veins and petioles contained measurable pyruvate concentrations in the range of 2 mM. The leaf vein form of the enzyme was purified approximately 143-fold, and, at the optimum pH of 5.6, the K_m value for pyruvate was 42 μM. This K_m is lower than the typical millimolar range seen for PDCs from other sources. The purified leaf PDC also decarboxylates 2-ketobutyric acid (K_m = 2.2 mM). We conclude that there are several possible sources of acetaldehyde production in cottonwood leaves: the well-characterized root-derived ethanol oxidation by ADH in leaves, and the decarboxylation of pyruvate by PDC in leaf veins, petioles, and other leaf tissues. Significantly, the leaf vein form of PDC with its high affinity for pyruvate, could function to shunt pyruvate carbon to the pyruvate dehydrogenase by-pass and thus protect the metabolically active vascular bundle cells from the effects of oxygen deprivation.     

Vanillate hydroxylase from the white rot basidiomycete Phanerochaete chrysosporium

A soluble enzyme fraction from Phanerochaete chrysosporium catalyzed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone. The enzyme, partially purified by ammonium sulfate precipitation, required NADPH and molecular oxygen for activity. NADH was not effective. Optimal activity was displayed between pH 7.5–8.5. Neither EDTA, KCN, NaN₃, nor o-phenanthroline (5 mM) were inhibitory. The enzyme was inducible with maximal activity displayed after incubation of previously grown cells with 0.1% vanillate for 30h.Abbreviations MHQ Methoxy-p-hydroquinone - GLC gas liquid chromatography - TMSi trimethylsilane - TLC thin layer chromatography     

Cloning, sequencing, and expression in Escherichia coli of the Bacillus pumilus gene for ferulic acid decarboxylase.

The Bacillus pumilus gene encoding a ferulic acid decarboxylase (fdc) was identified and isolated by its ability to promote ferulic acid decarboxylation in Escherichia coli DH5 alpha. The DNA sequence of the fdc gene was determined, and the recombinant enzyme produced in E. coli was purified and characterized.     

Purification and characterization of a new NAD(+)-dependent enzyme, L-tartrate decarboxylase, from Pseudomonas sp. group Ve-2.

A new enzyme, L-tartrate decarboxylase, was found in cells of Pseudomonas sp. group Ve-2. The enzyme was purified to homogeneity and characterized. The enzyme requires K+, Mg2+, and NAD+ for L-tartrate decarboxylation. The dependence of the enzymatic decarboxylation on NAD+ suggests that the decarboxylation involves redox reactions of the substrate. The enzyme catalyzes NAD(+)-linked oxidative decarboxylation of D-malate as well. The enzyme is composed of four subunits with identical molecular weight (Mr 40,000). The apparent Michaelis constants for L-tartrate and NAD+ are 1.1 mM, respectively. The cofactor requirements and the physical properties of the enzyme were similar to those of L-tartrate dehydrogenase-D-malate dehydrogenase from Rhodopseudomonas sphaeroides, and tartrate dehydrogenase from P. putida.     

6-Phosphogluconate dehydrogenase from Drosophila melanogaster. I. Purification and properties of the a isozyme

6-Phosphogluconate dehyrogenase is evident at all developmental stages of Drosophila melanogaster. The activity level is highest in early third instar larvae and declines to a lower, but relatively constant, level at all later stages of development. The enzyme is localized in the cytosolic portion of the cell. The A-isozymic form of 6-phosphogluconate dehydrogenase was purified to homogeneity and has a molecular weight of 105,000. The enzyme is a dimer consisting of subunits with molecular weights of 55,000 and 53,000. For the oxidative decarboxylation of 6-phosphogluconate the K_m for substrate is 81 µm while that for NADP⁺ is 22.3 µm. The optimum pH for activity is 7.8 while the optimum temperature is 37 C.This work was supported by National Research Council of Canada Grant A5860 and by the University of Calgary Research Policy and Grants Committee.     

A novel mechanism involved in the metabolism of the tartaric acid stereoisomers in Rhodopseudomonas sphaeroides: enzymatic conversion of meso-tartaric acid to D(-)-glyceric acid and CO2

In cell extracts of Rhodopseudomonas sphaeroides grown on meso-tartrate the activities of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and 1.1.1.83, respectively) could be measured spectrophotometrically but not the activity of a meso-tartrate dehydrogenase or dehydratase. However, an enzyme activity was detected manometrically that catalyzed the stoichiometric release of CO₂ from mesotartrate in a molar ratio of 1:1. This reaction required catalytic amounts of NAD and the presence of both divalent (Mn²⁺ or Mg²⁺) and monovalent (NH ₄ ⁺ or K⁺) cations. Purification of the meso-tartrate decarboxylase showed that it was part of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating), which thus possessed a third catalytic function. The homogeneous enzyme catalyzed the stoichiometric conversion of incso-tartaric acid to D(-)-glyceric acid and CO₂. All interfering catalytic activities had been eliminated during the course of enzyme purification.     

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The K _m-value of 1 M for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with ¹⁸O₂ and H₂ ¹⁸O indicated the incorporation of ¹⁸O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).Non-standard abbreviations DPE diphenylether - 2,3-dihydroxy-DPE 2,3-dihydroxydiphenylether - PCA 2-pyrone-6-carboxylic acid - 2,3-dihydroxy-BP dioxygenase 2,3-dihydroxybiphenyl dioxygenase - GC gas chromatography     

A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.