N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Production of ammonium dependent on basicL-amino acids byAnacystic nidulans

The incubation of the cyanobacteriumAnacystis nidulans withL-Arg,L-Lys orL-Orn, but neither with the correspondingD-isomers nor with other twentyL-amino acids, resulted in the production of large amounts of ammonium which accumulated in the outer medium. Relevant properties of thisin vivo ammonium production activity have been studied in cell suspensions treated with the glutamine synthetase inactivatorL-methionine-D,l-sulfoximine (MSX) to prevent assimilation by the cells of the resulting ammonium. In addition to its specificity for the basicL-amino acids, the system exhibited a set of properties (K _m value for substrates, requirement of oxygen which is taken up stoichiometrically with the production of ammonium, inhibition by o-phenanthroline and divalent cations) all of which are shared by a peculiarL-amino acid oxidase recently isolated fromA. nidulans. The data strongly suggest the participation of this enzyme in the production of ammonium from basic amino acids byA. nidulans, an activity that could account for the ability of this cyanobacterium to use arginine as a nitrogen source.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanide p-trifluoromethoxy-phenylhydrazone - MSX L-methionine-D,l-sulfoximine     

α-Chymotrypsin-Catalyzed Peptide Synthesis Using <Emphasis Type="Italic">N</Emphasis>-Protected <Emphasis Type="SmallCaps">D</Emphasis>-Amino Acid Carbamoylmethyl Esters as Acyl Donors

α-Chymotrypsin-catalyzed peptide synthesis was carried out between an N-protected D-amino acid ester and an L-amino acid amide (acyl donor, 10 mM; acyl acceptor, 50 mM; enzyme, 2 mg ml⁻¹; pH 8). By using a highly reactive carbamoylmethyl (Cam) ester as acyl donor, the D-amino acid was incorporated into the N-terminus of the resulting dipeptide amide. N-Protected dipeptide amides bearing D-amino acids such as D-Phe, D-Leu and D-Ala at their N-terminus were synthesized in high yields (up to 80%) in 1–3 h.     

Chemical composition of Eubacterium nodatum cell wall peptidoglycan

The structure of Eubacterium nodatum cell wall peptidoglycan was investigated. The peptide subunit of E. nodatum peptidoglycan has the following structure: L-Ala-D-Glu (Gly)-L-Orn-D-Ala. The carboxyl group of alanine occupying position 4 is attached to the -amino group of ornithine of an other subunit by the cross-linking bridge L-Ala-L-Ala-L-Orn. All glycine molecules are connected with the -carboxyl group of glutamic acid with the ratio being 0.5–1. The hydrolysis of E. nodatum peptidoglycan by the S. albus G enzyme proceeds primarily due to the activity of alanyl-alanine endopeptidase, ornithyl-ornithine endopeptidase, ornithyl-alanine endopeptidase, N-acetyl-muramyl-alanine amidase, N-acetylmuramidase and N-acetylglucosaminidase.     

Biochemical characterization of glutamine synthetase from the diazotrophic cyanobacterium,Anabaena doliolum

In cyanobacteria, the glutamine synthetase-L-glutamine-2-oxoglutarate aminotransferase (GS-GOGAT) pathway is the major ammonia-assimilating route. The GS ofAnabaena doliolum was synthesized more under N₂-fixing conditions, followed by ammonium, nitrate, and nitrite as nitrogen sources. The activities of both the glutamine synthetase, Mg²⁺-dependent biosynthetic and Mn²⁺-dependent -glutamyl transferase were optimum at pH 7. The active site of the enzyme bears sulfhydryl (-SH) groups; this was confirmed with the-SH group inhibitors, para-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM). The biosynthetic and -glutamyl transferase activities showed specificity for the divalent cations, Mg²⁺ and Mn²⁺, respectively. The other divalent cations Co²⁺, Cu²⁺, and Ni²⁺ were poor substitutes. This enzyme also required these divalent cations to stabilize its structure and function under extreme conditions such as high and low temperatures and urea denaturation. The glutamate analogl-methionine-d,l-sulfoximine, inactivated the enzyme, whereas the GOGAT inhibitor, azaserine, had no effect on the enzyme activity. The GS enzyme required de novo protein synthesis.     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Metabolism of α-aminoisobutyric acid in mungbean hypocotyls in relation to metabolism of 1-aminocyclopropane-1-carboxylic acid

1-Aminocyclopropane-1-carboxylic acid (ACC) is known to be converted to ethylene and conjugated into N-malonyl-ACC in plant tissues. When -amino[1-¹⁴C]isobutyric acid (AIB), a structural analog of ACC, was administered to mungbean (Vigna radiata L.) hypocotyl segments, it was metabolized to ¹⁴CO₂ and conjugated to N-malonyl-AIB (MAIB). -Aminoisobutyric acid inhibited the conversion of ACC to ethylene and also inhibited, to a lesser extent, N-malonylation of ACC and d-amino acids. Although the malonylation of AIB was strongly inhibited by ACC as well as by d-amino acids, the metabolism of AIB to CO₂ was inhibited only by ACC but not by d-amino acids. Inhibitors of ACC conversion to ethylene such as anaerobiosis, 2,4-dinitrophenol and Co²⁺, similarly inhibited the conversion of AIB to CO₂. These results indicate that the malonyalation of AIB to MAIB is intimately related to the malonylation of ACC and d-amino acids, whereas oxidative decarboxylation of AIB is related to the oxidative degradation of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyric acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid - MAIB -(malonylamino)-isobutyric acid - Mes 2-(N-morpholino)ethanesulfonic acid     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

Competitive reactions of a ruthenium arene anticancer complex with histidine, cytochrome c and an oligonucleotide

The ruthenium arene anticancer complex [(⁶-bip)Ru(en)Cl][PF₆] (1) (bip is biphenyl, en is ethylenediamine) reacted slowly with the amino acid L-histidine (L-His) in aqueous solution at 310 K. Two L-His adducts of 1 were separated by high-performance liquid chromatography and identified by electrospray ionisation mass spectrometry and NMR: an imidazole N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺, and an N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺. At 310 K, after 24 h only about 22% of complex 1 (2 mM) reacted with L-His, and of the unreacted 1, 59% had hydrolysed. In the presence of 100 mM NaCl, approximately 90% of 1 remained unreacted. In aqueous solution or triethylammonium acetate (TEAA) buffer (pH 7.6), ¹⁵N-labelled 1 reacted with cytochrome c to give two monoruthenated protein adducts. The reaction reached equilibrium within 2 h by which time approximately 50% of cytochrome c was ruthenated. On the basis of [¹H, ¹⁵N] NMR data, one adduct may have Ru bound to the N-terminus, and the other to a carboxylate group on the protein. In TEAA buffer and at 310 K, more than 90% of the 14-mer oligonucleotide d(TATGTACCATGTAT) reacted with 2 mol Eq of 1 to give rise to monoruthenated and diruthenated oligonucleotide adducts. The presence of cytochrome c (1 mol Eq) or L-His (4 mol Eq) had little effect on the course of the reaction with the oligonucleotide. In cells, DNA (or RNA) may be a favoured reaction site for this Ru anticancer complex.Electronic supplementary material is available for this article at .

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Lysine degradation in Candida maltosa: occurrence of a novel enzyme,acetyl-CoA: L-lysine N-acetyltransferase

The yeast Candida maltosa can utilize L-lysine as sole nitrogen and sole carbon source accompanied by accumulation of -N-acetyl-L-lysine, indicating that lysine is metabolized by way of N-acetylated intermediates. A novel lysine acetyltransferase catalyzing the first step in this pathway, the N-acetylation of the -amino group of L-lysine, was found in this yeast. The enzyme, acetyl-CoA:L-lysine N-acetyltransferase, is strongly induced in cells grown on L-lysine as sole carbon source. The enzyme is specific for both L-lysine and acetyl-CoA. The K _m values are 10 mM for L-lysine and 0.33 mM for acetyl-CoA. The enzyme has a maximum activity at pH 8.1.Dedicated to Prof. Dr. F. Böttcher in occasion of his 60th birthday     

Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis

Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected. Using these mutants we could determine two functional roles for the L-leucine dehydrogenase. This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source. Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain -keto acids which are precursors of branched chain fatty acid biosynthesis. These results together with the inducibility of the enzyme suggest that the L-leucine dehydrogenase has primarily a catabolic rather than an anabolic function in the metabolism of Bacillus subtilis.     

Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus

Leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD⁺ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of -ketoisocaproate and -ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD⁺ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei

Summary The new enzyme d-2-hydroxyisocaproate dehydrogenase (NAD⁺-dependent) was detected in strains of the genus Lactobacillus and related genera. Straight and branched chain aliphatic as well as aromatic 2-ketocarboxylic acids are stereospecifically reduced to the corresponding d-2-hydroxycarboxylic acids according to the following equation:R-CO-COOH + NADH + H⁺ R-CHOH-COOH + NAD⁺ The enzyme is called d-hydroxyisocaproate dehydrogenase by us because 2-ketoisocaproate is the substrate with the lowest K_M-value. NAD(H) as a cofactor cannot be replaced by NADP(H). Because of its broad substrate specificity we chose the strain Lactobacillus casei ssp. pseudoplantarum (DSM 20 008) for enzyme production and characterization. d-2-hydroxyisocaproate dehydrogenase could be purified 180-fold starting with 500 g of wet cells.The purification procedure involved liquid-liquid extraction with aqueous two-phase systems and ion-exchange chromatography. At this stage the enzyme has a specific activity of 25 U/mg and can be used for technical applications. Further purification up to a homogeneous protein with a specific activity of 110 U/mg can be achieved by chromatography on Amberlite CG 50 at pH 3.5. Properties important for technical application of the d-HicDH were investigated, especially the substrate specificity and the optimum pH- and temperature ranges for activity and stability of the catalist.     

Purification and properties of a novel enzyme,N-acylamino acid racemase,from Streptomyces atratus Y-53

A novel enzyme, N-acylamino acid racemase, was purified to homogeneity from Streptomyces atratus Y-53 and characterized. This enzyme catalyzes the interconversion of optically active N-acylamino acids. The relative molecular mass (M_r) of the enzyme was estimated to be about 41 000 and 244 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme is composed of six subunits with an equal M_r. The enzyme showed a broad substrate specificity toward N-acylamino acids, such as N-acetylmethionine, N-chloroacetylphenylalanine and N-chloroacetylvaline. The apparent Michaelis constant (K_m) values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 15.2 and 5.6 mm, respectively. Enzyme activity was markedly enhanced by divalent metal ions, such as Co²⁺, Mg²⁺ and Mn²⁺, and was inhibited by metal-chelating reagent, indicating that the enzyme is a metalloenzyme. We propose to name the enzyme N-acylamino acid racemase (acylamino acid racemase). Correspondence to: S. Tokuyama     

Polymyxin Acylase: An Enzyme Causing Intramolecular N2⇄N6 Acyl Transfer in N-Monooctanoyl-l-Lysine

Polymyxin acylase from Pseudomonas sp. M-6-3 can deacylate not only polymyxin antibiotics, but also A-fatty acyl-peptides and N-fatty acyl-amino acids. We found that this enzyme causes intramolecular N²?N⁶ acyl transfer in monooctanoyl-l-lysine; when N²-octanoyl-l-lysine is the substrate, N⁶-octanoyl- l-lysine is produced at pH 10.5, but when N⁶-octanoyl- l-lysine is the substrate, N²-octanoyl- l-lysine is produced at pH 8.0. In these reactions, the deacylation proceeded gradually at the final stage and eventually, both N²-octanoyl- l-lysine and N⁶-octanoyl- l-lysine were hydrolyzed to l-lysine and octanoic acid. Furthermore, this enzyme showed intermolecular acyltrans- ferase activity, transferring several N-octanoyl- dl-amino acids to N-octanoyl-hydroxylamine. This acyltransfer ability of polymyxin acylase offers a new method of enzymic N-acylation of compounds containing amino components.     

Purification and characterization of dihydroorotase fromPseudomonas putida

Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK _m andV _max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min⁻¹ mg⁻¹, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min⁻¹ mg⁻¹, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn²⁺. However, excessive Zn²⁺ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK _i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK _i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.     

L-Phenylalanine formation from acetamidocinnamic acid by newly isolated bacteria

Summary Bacteria with the ability to form L-phenylalanine from acetamidocinnamic acid were isolated from several soils. Among them, strain no. S-7 and strain no. N-7 were identified as Alcaligenus faecalis S-7 and Bacillus sphaericus N-7, respectively. The L-phenylalanine-forming enzyme systems in both bacteria were found to be inducible and intracellular. With intact cells of both bacteria and 40 mg/ml as wet base, 10 mg/ml acetamidocinnamic acid was utilized, and 7.7 mg/ml L-phenylalanine in a molar yield of 94% was produced after 72h incubation. The pathway of L-phenylalanine formation is considered to take the following course: acetamidocinnamic acid is deacetylated to -amino cinnamic acid; this is spontaneously changed to phenylpyruvic acid, and L-phenylalanine is formed by transamination.     

D-amino acid N-acetyltransferase of Saccharomyces cerevisiae: a close homologue of histone acetyltransferase Hpa2p acting exclusively on free D-amino acids

d-Amino acid N-acetyltransferase is a unique enzyme of Saccharomyces cerevisiae acting specifically on d-amino acids. The enzyme was found to be encoded by HPA3, a putative histone/protein acetyltransferase gene, and we purified its gene product, Hpa3p, from recombinant Escherichia coli cells. Hpa3p shares 49% sequence identity and 81% sequence similarity with a histone acetyltransferase, Hpa2p, of S. cerevisiae. Hpa3p acts on a wide range of d-amino acids but shows extremely low activity toward histone. However, Hpa2p does not act on any of the free amino acids except l-lysine and d-lysine. Kinetic analyses suggest that Hpa3p catalyzes the N-acetylation of d-amino acids through an ordered bi-bi mechanism, in which acetyl-CoA is the first substrate to be bound and CoA is the last product to be liberated.     

Purification,characterization, gene cloning and nucleotide sequencing of D-stereospecific amino acid amidase from soil bacterium: Delftia acidovorans

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40°C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45°C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd²⁺, Ag⁺, Zn²⁺, Hg²⁺ and As³⁺ . The NH₂ terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.

     

Taurine Levels in Some Tissues of Yellowtail (Seriola quinqueradiata) and Mackerel (Scomber japonicus)

The mechanism of stereospecific production of l-amino acids from the corresponding 5-substituted hydantoins by Bacillus brevis AJ-12299 was studied. The enzymes involved in the reaction were partially purified by DEAE-Toyopearl 650M column chromatography and their properties were investigated. The conversion of dl-5-substituted hydantoins to the corresponding l-amino acids consisted of the following two successive reactions. The first step was the ring-opening hydrolysis to N-carbamoyl amino acids catalyzed by an ATP dependent l-5-substituted hydantoin hydrolase. This reaction was stereospecific and the N-carbamoyl amino acid produced was exclusively the l-form. N-Carbamoyl-l-amino acid was also produced from the d-form of 5-substituted hydantoin, which suggests that spontaneous racemization occurred in the reaction mixture. In the second step, N-carbamoyl-l-amino acid was hydrolyzed to l-amino acid by an N-carbamoyl-l-amino acid hydrolase, which was also an l-specific enzyme. The ATP dependency of the l-5-substituted hydantoin hydrolase was supposed to be the limiting factor in the production of l-amino acids from the corresponding 5-substituted hydantoins by this bacterium.