High-performance liquid chromatographic determination of lipoamidase (lipoyl-X hydrolase) activity with a novel substrate, lipoyl-6-aminoquinoline

An HPLC lipoamidase (lipoyl-X hydrolase) assay method has been developed, which uses a novel fluorescent substrate, lipoyl-6-aminoquinoline (LAQ). LAQ is synthesized from lipoic acid and 6-aminoquinoline (AQ) through lipoyl chloride as an intermediate and is conveniently purified by washing with chloroform-methanol. Mechanistic studies on the time-course, the dependence on enzyme and substrate concentrations were performed by using LAQ and a model enzyme (milk lipoamidase). Moreover, this method was successfully applied to the direct determination of the lipoamidase (LAQ hydrolase) activity in samples of human liver, milk, stools and porcine serum. Using this novel synthetic lipoyl substrate, we demonstrated that LAQ hydrolase was present in some specific tissues: LAQ hydrolase was solely present in the grey matter and not in the white matter in the human cerebrum. Furthermore, LAQ hydrolase activity was shown to increase in human liver cancer. Thus, this enzyme assay method is expected to be applicable to the tissue distribution study and also to the basic research on human diseases such as cancer.     

Human placental glutamate dehydrogenase - purification - kinetic and regulatory properties - physicochemical studies

Glutamate dehydrogenase (EC. 1.4.1.3) has been purified more than 9,000 times from human placental alcoholic subfractions as a homogenous protein of 55,155 daltons (subunit molecular weight). Kinetic constants for the reverse reaction (reductive amination of α-ketoglutarate) have been shown to be similar to those of the bovine liver enzyme, while the kinetic constants for the forward reaction were markedly different as well as some regulatory properties (lack of activation by ADP in the reverse reaction). The amino acid composition differs from the bovine liver enzyme composition. Furthermore, the tryptic peptide patterns of the placental enzyme and the human liver enzyme have been compared. Besides the low specific activity of this enzyme, the results indicate that human placental glutamate dehydrogenase is closely related to other mammalian glutamate dehydrogenases.     

Human liver alcohol dehydrogenase: purification, composition, and catalytic features.

Alcohol dehydrogenase has been purified from human liver by affinity chromatography. Ultracentrifugation, Sephadex G-200 chromatography, and amino acid analyses of multiple preparations demonstrate homogeneity of molecular weight. Sodium dodecyl sulfate disc gel electrophoresis reveals a single species of molecular weight 42 000. Based on a molecular weight of 85 000 for the dimer obtained from the amino acid composition and a molar absorptivity of A280nm0.1% = 0.58, the enzyme contains 3.6-4.2 g-atoms of zinc, as determined by emission spectrography, microwave-induced emission, and atomic absorption spectrometry. Inhibition by o-phenanthroline, (ethylenedinitrilo)tetraacetic acid, and alpha,alpha'-bipyridine demonstrates that zinc is essential to enzymatic function. Detailed kinetic analyses using primary alcohols of the homologous series CH3(CH2)nOH, n = 0-5, and the corresponding aldehydes as substrates show that KM values become smaller as n increases. This suggest that hydrophobic interactions play a role in substrate binding. The availability of well-defined preparations of human liver alcohol dehydrogenase now allows definitive genetic and functional studies of this enzyme to elucidate human ethanol metabolism.     

On the iron-sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase.

Porcine liver dihydropyrimidine dehydrogenase is a homodimeric iron-sulfur flavoenzyme that catalyses the first and rate-limiting step of pyrimidine catabolism. The enzyme subunit contains 16 atoms each of nonheme iron and acid-labile sulfur, which are most likely arranged into four [4Fe-4S] clusters. However, the presence and role of such Fe-S clusters in dihydropyrimidine dehydrogenase is enigmatic, because they all appeared to be redox-inactive during absorbance-monitored titrations of the enzyme with its physiological substrates. In order to obtain evidence for the presence and properties of the postulated four [4Fe-4S] clusters of dihydropyrimidine dehydrogenase, a series of EPR-monitored redox titrations of the enzyme under a variety of conditions was carried out. No EPR-active species was present in the enzyme 'as isolated'. In full agreement with absorbance-monitored experiments, only a small amount of neutral flavin radical was detected when the enzyme was incubated with excess NADPH or dihydrouracil under anaerobic conditions. Reductive titrations of dihydropyrimidine dehydrogenase with dithionite at pH 9.5 and photochemical reduction at pH 7.5 and 9.5 in the presence of deazaflavin and EDTA led to the conclusion that the enzyme contains two [4Fe-4S]2+,1+ clusters, which both exhibit a midpoint potential of approximately -0.44 V (pH 9.5). The two clusters are most likely close in space, as demonstrated by the EPR signals which are consistent with dipolar interaction of two S = 1/2 species including a half-field signal around g approximately 3.9. Under no circumstances could the other two postulated Fe-S centres be detected by EPR spectroscopy. It is concluded that dihydropyrimidine dehydrogenase contains two [4Fe-4S] clusters, presumably determined by the C-terminal eight-iron ferredoxin-like module of the protein, whose participation in the enzyme-catalysed redox reaction is unlikely in light of the low midpoint potential measured. The presence of two additional [4Fe-4S] clusters in dihydropyrimidine dehydrogenase is proposed based on thorough chemical analyses on various batches of the enzyme and sequence analyses. The N-terminal region of dihydropyrimidine dehydrogenase is similar to the glutamate synthase beta subunit, which has been proposed to contain most, if not all, the cysteinyl ligands that participate in the formation of the [4Fe-4S] clusters of the glutamate synthase holoenzyme. It is proposed that the motif formed by the Cys residues at the N-terminus of the glutamate synthase beta subunit, which are conserved in dihydropyrimidine dehydrogenase and in several beta-subunit-like proteins or protein domains, corresponds to a novel fingerprint that allows the formation of [4Fe-4S] clusters of low to very low midpoint potential.     

The purification and characterization of glutaryl-coenzyme A dehydrogenase from porcine and human liver

Glutaryl-CoA dehydrogenase, a multifunctional enzyme responsible for dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA, has been purified 1,680-fold from porcine liver mitochondria. The purified porcine enzyme has a subunit molecular weight of 47,800 and a native molecular weight of 190,500. Porcine glutaryl-CoA dehydrogenase catalyzed the conversion of [1,5-14C]glutaryl-CoA to [14C] crotonyl-CoA and 14CO2 in a 1:1:1 ratio. The porcine enzyme has Km values for electron transfer flavoprotein and glutaryl-CoA of 1.1 and 3.3 microM, respectively, and turnover numbers of 860 mol of electron transfer flavoprotein/min/mol of glutaryl-CoA dehydrogenase and 327 mol of glutaryl-CoA/min/mol of glutaryl-CoA dehydrogenase. Human glutaryl-CoA dehydrogenase has been purified 1,278-fold from human liver mitochondria. The purified human enzyme has a subunit molecular weight of 58,800 and a native molecular weight of 256,000. Human glutaryl-CoA dehydrogenase showed a reaction of only partial identity when compared to porcine glutaryl-CoA dehydrogenase by Ouchterlony double immunodiffusion analysis using antiserum raised against and monospecific for porcine glutaryl-CoA dehydrogenase.     

Purification and properties of the bovine liver mitochondrial dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase has been purified 6,000-fold from bovine liver mitochondria to apparent homogeneity in six steps. Electrophoretic migration of the homogeneous enzyme on sodium dodecyl sulfate-polyacrylamide gels reveals a subunit Mr of 42,000. By contrast to the well-characterized, cytosolic dihydroorotate oxidases (EC 1.3.3.1), the purified bovine dehydrogenase is a dihydroorotate:ubiquinone oxidoreductase. Maximal rates of orotate formation are obtained using coenzymes Q6 or Q7 as cosubstrate electron acceptors. Concomitant with substrate oxidation, the enzyme will reduce simple quinones, such as benzoquinone, but at significantly lower rates (10-15%) than that obtained for reduction of coenzyme Q6. Enzyme-catalyzed substrate oxidation is not supported by molecular oxygen. The specificity of the purified enzyme for dihydropyrimidine substrates has also been explored. The methyl-, ethyl-, t-butyl-, and benzyl-S-dihydroorotates are substrates, but 1- and 3-methyl and 1,3-dimethyl methyl-S-dihydroorotates are not. Competitive inhibitors include product orotate, 5-methyl orotate, and racemic cis-5-methyl dihydroorotate.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.     

Purification and characterization of N-methylalanine dehydrogenase.

Cell free extracts of Pseudomonas MS previously have been shown to carry out the synthesis of a novel amino acid, N-methylalanine (Kung, H.F., and Wagner, C. (1970) Biochim. Biophys. Acta 201, 513-516). An enzyme has been isolated from this organism which is responsible for the synthesis of N-methylalanine. The stoichiometry of the reaction catalyzed by this enzyme leads to the following formulation: Methylamine + pyruvate + NADPH + H-+ yields N-methylalanine + NADP-+ + H2O. This enzyme has been physically separated from alanine dehydrogenase, which is also present in these extracts. This new enzyme has been named N-methylalanine dehydrogenase. It has been purified to near homogeneity as judged by disc gel electrophoresis. Gel filtration chromatography showed that N-methylalanine dehydrogenase has an apparent molecular weight of 77,000, while electrophoresis in sodium dodecyl sulfate gave rise to a single band with a molecular weight of approximately 36,500. The enzyme is optimally active in the pH range between 8.2 and 8.6. The apparent K-m values for pyruvate, NADPH, and methylamine, respectively, are 1-5 times 10 minus 2 M, 3-5 times 10 minus 5 M, and 7.5 times 10 minus 2 M.     

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.     

Molecular differences between rat-liver and rat-kidney biliverdin reductase. Implications for their in vivo regulation

Rat-liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular mass of 34 kDa, while the minor form 2 has a molecular mass of 56 kDa. Form 1 was converted into a second major form (form 3) with a molecular mass of 68 kDa by a NAD+-dependent peroxisomal dehydrogenase which was induced under conditions of oxidative stress [Frydman, R. B., Tomaro, M. L., Awruch, J. & Frydman, B. (1984) Biochem. Biophys. Res. Commun. 121, 249]. Molecular form 1 from rat kidney was not affected by the dehydrogenase, and a structural explanation for this difference was therefore sought. Both form 1 biliverdin reductases, isolated from rat liver and kidney, were purified to homogeneity using affinity chromatography, FPLC and HPLC techniques. The homogeneous enzymes were found to be identical when compared by their HPLC retention times, amino acid compositions and electrophoretic behaviour on polyacrylamide gels under non-denaturing conditions and on SDS/polyacrylamide gels. On HPLC analysis the peptides resulting from the CNBr cleavage were found to be the same for both enzymes, when either the native enzymes or their thioethylpyridine derivatives were compared. When the HPLC fingerprints of the tryptic digests were compared, they were found to be very similar, except for a peptide eluting at 31.60 min in the liver digest and at 23.60 min in the kidney digest. When the enzyme from both origins was alkylated with 4-dimethylaminoazobenzene-4'-iodoacetamide and then digested with trypsin, the HPLC fingerprints of the alkylated cysteine-carrying peptides were almost identical, except for a peptide with a retention time of 19.03 min in the liver digest and of 18.19 min in the kidney digest. The liver reductase was not amenable to Edman degradation suggesting a block at the NH2-terminus; in the kidney enzyme, however, it was free and an NH2-terminal sequence of 12 amino acids could be determined. The liver enzyme was found to be more sensitive toward p-hydroxymercuriphenyl sulfonate than the kidney enzyme.     

Erythrocyte acid phosphatase: species specificity in activity modulation by purine analogs

The previous studies of the interaction of purine analogs and human erythrocyte acid phosphatase isozymes were extended to include erythrocyte acid phosphatase from seven other species. Consistent responses, similar to the observations with the several genotypically different human isozymes, were observed. The isozyme from chimpanzee erythrocytes was similar to the human B-type isozyme while the baboon and cow isozymes were at the other extreme in responsiveness and were more divergent from the B-isozyme than was the human A-type isozyme. The ACP from rabbit, dog, sheep and rhesus erythrocytes exhibited intermediate levels of responsiveness but did differ from the human A-type isozyme. Additional studies indicated some differences between the responsiveness of the partially purified erythrocyte enzyme and the low molecular weight ACP from liver.     

Purification and characterization of a rat brain aldehyde dehydrogenase able to metabolize gamma-aminobutyraldehyde to gamma-aminobutyric acid.

An enzyme which catalyses dehydrogenation of gamma-aminobutyraldehyde (ABAL) to gamma-aminobutyric acid (GABA) was purified to homogeneity from rat brain tissues by using DEAE-cellulose and affinity chromatography on 5'-AMP-Sepharose, phosphocellulose and Blue Agarose, followed by gel filtration. Such an enzyme was first purified from mammalian brain tissues, and was identified as an isoenzyme of aldehyde dehydrogenase. It has an Mr of 210,000 determined by polyacrylamide-gradient-gel electrophoresis, and appeared to be composed of subunits of Mr 50,000. The close similarity of substrate specificity toward acetaldehyde, propionaldehyde and glycolaldehyde between the enzyme and other aldehyde dehydrogenases previously reported was observed. But substrate specificity of the enzyme toward ABAL was higher than those of aldehyde dehydrogenases from human liver (E1 and E2), and was lower than those of ABAL dehydrogenases from human liver (E3), Escherichia coli and Pseudomonas species. The Mr and relative amino acid composition of the enzyme are also similar to those of E1 and E2. The existence of this enzyme in mammalian brain seems to be related to a glutamate decarboxylase-independent pathway (alternative pathway) for GABA synthesis from putrescine.     

Isolation and characterization of an aldehyde dehydrogenase encoded by the aldB gene of Escherichia coli

An aldehyde dehydrogenase was detected in crude cell extracts of Escherichia coli DH5alpha. Growth studies indicated that the aldehyde dehydrogenase activity was growth phase dependent and increased in cells grown with ethanol. The N-terminal amino acid sequence of the purified enzyme identified the latter as an aldehyde dehydrogenase encoded by aldB, which was thought to play a role in the removal of aldehydes and alcohols in cells that were under stress. The purified enzyme showed an estimated molecular mass of 220 +/- 8 kDa, consisting of four identical subunits, and preferred to use NADP and acetaldehyde. MgCl2 increased the activity of the NADP-dependent enzyme with various substrates. A comparison of the effect of Mg2+ ions on the bacterial enzyme with the effect of Mg2+ ions on human liver mitochondrial aldehyde dehydrogenase revealed that the bacterial enzyme shared kinetic properties with the mammalian enzyme. An R197E mutant of the bacterial enzyme appeared to retain very little NADP-dependent activity on acetaldehyde.     

Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.

Human erythrocyte aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) was purified to apparent homogeneity. The native enzyme has a molecular weight of about 210,000 as determined by gel filtration, and SDS-polyacrylamide gel electrophoresis of this enzyme yields a single protein and with a molecular weight of 51,500, suggesting that the native enzyme may be a tetramer. The enzyme has a relatively low Km for NAD (15 microM) and a high sensitivity to disulfiram. Disulfiram inhibits the enzyme activity rapidly and this inhibition is apparently of a non-competitive nature. In kinetic characteristic and sensitivity to disulfiram, erythrocyte aldehyde dehydrogenase closely resembles the cytosolic aldehyde dehydrogenase found in the liver of various species of mammalians.     

A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.     

First purification of the antiquitin protein and demonstration of its enzymatic activity

Antiquitin is an evolutionarily conserved protein believed to play a role in the regulation of cellular turgor. Based on sequence analysis, this protein is classified as a member of the aldehyde dehydrogenase superfamily. All previous studies on antiquitin have been confined to the nucleotide level, and the protein has never been purified and characterized. In the present investigation, the antiquitin protein was purified for the first time. An acetaldehyde-oxidizing protein was isolated from the liver of black seabream (Mylio macrocephalus) by chromatographies on alpha-cyanocinnamate Sepharose and Affi-gel Blue agarose, followed by ammonium sulfate precipitation. The purified protein was identified as antiquitin by the first 18 N-terminal amino acid residues which showed 83.3% identity with the deduced sequence of human antiquitin. Electrophoretic mobility studies indicated that black seabream antiquitin is a tetramer with a subunit molecular mass of 57.5 kDa. Kinetic analysis of the purified protein indicated that it catalyzes the oxidation of acetaldehyde with K(m) and V(max) values of 2.0 mM and 1.3 U/mg, respectively. The longer aliphatic propionaldehyde and the aromatic benzaldehyde are also substrates of the purified enzyme. The enzyme is highly specific towards NAD+ as the coenzyme and is totally inactive towards NADP+. Maximal enzymatic activity was found at about pH 9-10.     

Identification of histidyl peptide labeled by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate in an ADP regulatory site of glutamate dehydrogenase

Bovine liver glutamate dehydrogenase reacts covalently with 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of 1 mol reagent/mol enzyme subunit and loss of one of the two ADP sites of native enzyme [S. P. Batra and R. F. Colman, J. Biol. Chem. 261, 15565-15571 (1986)]. Incorporation of reagent is prevented specifically by ADP. The modified enzyme has now been digested with trypsin. The nucleotidyl peptide has been purified by chromatography on phenylboronate-agarose, followed by reverse-phase HPLC. On the basis of amino acid composition following acid hydrolysis, and gas-phase sequencing, the modified tryptic peptide was established as Ala-Gln-His-Ser-Gln-His-Arg, corresponding to amino acids 80-86 of the known glutamate dehydrogenase primary structure. The evidence presented indicates that the target amino acid attacked by 2-BDB-TAMP is histidine-82 and that this residue is located within the high-affinity ADP-activating site of glutamate dehydrogenase. In the course of this work, it was found that the positions of Gln84 and His85 had been reported as reversed in the revised sequence of bovine liver glutamate dehydrogenase [J. H. Julliard and E. L. Smith, J. Biol. Chem. 254, 3427-3438 (1979)]. Three additional corrections are here reported in the amino acid sequence of the native enzyme on the basis of gas-phase sequencing of other peptides purified by HPLC: Asp168 (not Asn); His221-Gly222 (not Gly-His); and Glu355 (not Gln).     

Stereospecificity of hepatic L-tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase [L-tryptophan--oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.     

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency is caused by mutations in the HADH2 gene

2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency is a novel inborn error of isoleucine degradation. In this article, we report the elucidation of the molecular basis of MHBD deficiency. To this end, we purified the enzyme from bovine liver. MALDI-TOF mass spectrometry analysis revealed that the purified protein was identical to bovine 3-hydroxyacyl-CoA dehydrogenase type II. The human homolog of this bovine enzyme is a short-chain 3-hydroxyacyl-CoA dehydrogenase, also known as the "endoplasmic reticulum-associated amyloid-beta binding protein" (ERAB). This led to the identification of the X-chromosomal gene involved, which previously had been denoted "HADH2." Sequence analysis of the HADH2 gene from patients with MHBD deficiency revealed the presence of two missense mutations (R130C and L122V). Heterologous expression of the mutant cDNAs in Escherichia coli showed that both mutations almost completely abolish enzyme activity. This confirms that MHBD deficiency is caused by mutations in the HADH2 gene.     

Purification and properties of 3 alpha-hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum from human intestine

3 alpha-Hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum, isolated from human intestinal bacteria, was capable of converting 3-ketoglycyrrhetic acid to 3 alpha-hydroxyglycyrrhetic acid. The enzyme was purified to homogeneity by means of butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A, Toyopearl HW-55S, and isoelectric focusing column chromatographies. The purified enzyme showed a specific activity of 156 mumol/min.mg toward 3 alpha-hydroxyglycyrrhetic acid, and showed a single band on SDS-polyacrylamide gel electrophoresis. The apparent molecular weight was 53,000, as estimated by gel filtration, and 30,000, as judged by SDS-polyacrylamide gel electrophoresis. Its isoelectric point was 5.2. The enzyme showed absolute specificity for the 3 alpha-hydroxyl and 3-ketonic groups of 18 alpha- or 18 beta-glycyrrhetic acid and required NADP+ and NADPH as cosubstrates. The enzyme did not act on any 3 alpha-hydroxyl or 3-ketonic group of steroids or bile acids. The enzyme is a novel type of enzyme, defined as 3 alpha-hydroxy-glycyrrhetinate dehydrogenase, being quite different from 3 alpha-hydroxysteroid dehydrogenase [EC 1.1.1.50].