Purification and properties of alpha-amino-epsilon-caprolactam racemase from Achromobacter obae.

We have purified a unique enzyme, alpha-amino-epsilon-caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl-Sepharose CL-4B and Thiopropyl-Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with Mr approximately 50000 and a sedimentation coefficient (S20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is approximately 9.0. D- and L-alpha-amino-epsilon-caprolactams are the only substrates. The Km values for the D- and L-isomers are, 8 and 6 mM, respectively.

Site-directed mutagenesis of the amino acid residues in beta-strand III [Val30-Val36] of D-amino acid aminotransferase of Bacillus sp. YM-1.

The beta-strand III formed by amino acid residues Val30-Val36 is located across the active site of the thermostable D-amino acid aminotransferase (D-AAT) from thermophilic Bacillus sp. YM-1, and the odd-numbered amino acids (Tyr31, Val33, Lys35) in the strand are revealed to be directed toward the active site. Interestingly, Glu32 is also directed toward the active site. We first investigated the involvement of these amino acid residues in catalysis by alanine scanning mutagenesis. The Y31A and E32A mutant enzymes showed a marked decrease in k(cat) value, retaining less than 1% of the wild-type enzyme activity. The k(cat) values of V33A and K35A were changed slightly, but the Km of K35A for alpha-ketoglutarate was increased to 35.6 mM, compared to the Km value of 2.5 mM for the wild-type enzyme. These results suggested that the positive charge at Lys35 interacted electrostatically with the negative charge at the side chain of alpha-ketoglutarate. Site-directed mutagenesis of the Glu32 residue was conducted to demonstrate the role of this residue in detail. From the kinetic and spectral characteristics of the Glu32-substituted enzymes, the Glu32 residue seemed to interact with the positive charge at the Schiff base formed between the aldehyde group of pyridoxal 5'-phosphate (PLP) and the epsilon-amino group of the Lys145 residue.

Thermostable alanine dehydrogenase of Bacillus sp. DSM730: gene cloning, purification, and characterization.

We have cloned the thermostable alanine dehydrogenase (EC 1.4.1.1) gene from a thermophile, Bacillus sp. DSM730, into Escherichia coli C600 with a vector plasmid, pBR322. The enzyme was overproduced by the transformed cells, and purified to homogeneity with a yield of 69% by heat treatment and another step. The enzyme has a molecular weight of about 250,000 and consists of 6 subunits identical in molecular weight (43,000). It is not inactivated by heat treatment at 75 degrees C for 60 min, or incubation in the pH range of 5.5-10.5 at 55 degrees C for 10 min. The enzyme ctalyzes the oxidative deamination of L-serine in addition to L-alanine. The oxo analogue of serine is as reactive as pyruvate. Thus, the enzyme differs markedly from alanine dehydrogenases so far studied.

Degradation of L-djenkolate catalyzed by S-alkylcysteine alpha,beta-lyase from Pseudomonas putida.

S-Alkylcysteine alpha, beta-lyase [EC 4.4.1.6] of Pseudomonas putida catalyzes alpha,beta-elimination of L-djenkolate [3,3'-methylenedithiobis(2-aminopropionic acid)] to produce pyruvate, ammonia, and S-(mercaptomethyl)cysteine initially. Secondly, S-(mercaptomethyl)-cysteine, which was identified in the form of S-(mercaptomethyl)cysteine thiolactone and S-(2-thia-3-carboxypropyl)cysteine in the absence and presence of iodoacetic acid, respectively, is decomposed enzymatically to pyruvate, ammonia, and bis(mercapto)methane, or spontaneously to cysteine, formaldehyde, and hydrogen sulfide. Balance studies showed that 1.3 mol each of pyruvate and ammonia and 0.2 mol each of formaldehyde and cysteine were produced with consumption of 1 mol of L-djenkolate. 1,2,4,5-Tetrathiane, 1,2,4-trithiolane, 1,2,4,6-tetrathiepane, and 1,2,3,5,6-pentathiepane, which are derivatives of bis(mercapto)methane, were also produced during the alpha,beta-elimination of L-djenkolate. In addition, a polymer with the general formula of -(CH2S)n- was produced as a white precipitate. When the alpha,beta-elimination of L-djenkolate was carried out in the presence of 20 mM iodoacetic acid, neither formaldehyde, cysteine, hydrogen sulfide, or the polymer were formed. Instead, the S-carboxymethyl derivatives of bis(mercapto)methane and S-(mercaptomethyl)cysteine were produced in addition to pyruvate and ammonia.

Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions.

We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine. When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine. Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine. Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine. We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected. This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization.

Mutation of arginine 98, which serves as a substrate-recognition site of D-amino acid aminotransferase, can be partly compensated for by mutation of tyrosine 88 to an arginyl residue.

D-Amino acid aminotransferase is the only aminotransferase that catalyzes the transamination of D-amino acids. We studied the role of the binding site for the alpha-carboxyl group of substrates, which is presumably crucial for the unique stereospecificity of the enzyme. The site-directed mutagenesis of Arg98, which is the putative carboxyl-binding site, as judged on the basis of X-ray crystallographic studies [Sugio, S., Petsko, G.A., Manning, J.M., Soda, K., and Ringe, D. (1995) Biochemistry 34, 9661-9669], by replacement with methionine and lysine, resulted in decreases in the kmax values and increases in the Kd values for both amino donors and amino acceptors. The introduction of another mutation, that of Tyr88, which is located near Arg98 in the spacial structure, by replacement with arginine, in addition to the above Arg98 mutation, resulted in increases in the kmax values but little change in the Kd values. These results suggest that Arg98 constitutes the carboxyl-binding site for the substrate, efficient catalysis by the enzyme being facilitated upon binding. The mutant enzymes are also relieved from inhibition by high concentrations of alpha-ketoglutarate, which is an inherent character of the wild-type enzyme. Therefore, Arg98 is also responsible for the inhibition by alpha-ketoglutarate.

Tyrosine 265 of alanine racemase serves as a base abstracting alpha-hydrogen from L-alanine: the counterpart residue to lysine 39 specific to D-alanine.

Alanine racemase of Bacillus stearothermophilus has been proposed to catalyze alanine racemization by means of two catalytic bases: lysine 39 (K39) abstracting specifically the alpha-hydrogen of D-alanine and tyrosine 265 (Y265) playing the corresponding role for the antipode L-alanine. The role of K39 as indicated has already been verified [Watanabe, A., Kurokawa, Y., Yoshimura, T., Kurihara, T., Soda, K., and Esaki, N. (1999) J. Biol. Chem. 274, 4189-4194]. We here present evidence for the functioning of Y265 as the base catalyst specific to L-alanine. The Y265-->Ala mutant enzyme (Y265A), like Y265S and Y265F, was a poor catalyst for alanine racemization. However, Y265A and Y265S catalyzed transamination with D-alanine much more rapidly than the wild-type enzyme, and the bound coenzyme, pyridoxal 5'-phosphate (PLP), was converted to pyridoxamine 5'-phosphate (PMP). The rate of transamination catalyzed by Y265F was about 9% of that by the wild-type enzyme. However, Y265A, Y265S, and Y265F were similar in that L-alanine was inert as a substrate in transamination. The apo-form of the wild-type enzyme catalyzes the abstraction of tritium non-specifically from both (4'S)- and (4'R)-[4'-(3)H]PMP in the presence of pyruvate. In contrast, apo-Y265A abstracts tritium virtually from only the R-isomer. This indicates that the side-chain of Y265 abstracts the alpha-hydrogen of L-alanine and transfers it supra-facially to the pro-S position at C-4' of PMP. Y265 is the counterpart residue to K39 that transfers the alpha-hydrogen of D-alanine to the pro-R position of PMP.

Role of tyrosine 265 of alanine racemase from Bacillus stearothermophilus.

Tyrosine 265 (Y265) of Bacillus stearothermophilus is believed to serve as a catalytic base specific to the L-enantiomer of a substrate amino acid by removing (or returning) an alpha-hydrogen from (or to) the isomer on the basis of the X-ray structure of the enzyme [Stamper, C.G., Morollo, A.A., and Ringe, D. (1998) Biochemistry 37, 10438-10443]. We found that the Y265-->Ala mutant (Y265A) enzyme is virtually inactive as a catalyst for alanine racemization. We examined the role of Y265 further with beta-chloroalanine as a substrate with the expectation that the Y265A mutant only catalyzes the alpha,beta-elimination of the D-enantiomer of beta-chloroalanine. However, L-beta-chloroalanine also served as a substrate; this enantiomer was rather better as a substrate than its antipode. Moreover, the mutant enzyme was as equally active as the wild-type enzyme in the elimination reaction. These findings indicate that Y265 is essential for alanine racemization but not for beta-chloroalanine elimination.

Reconsideration of the essential role of a histidine residue of L-2-halo acid dehalogenase.

His20 of L-2-halo acid dehalogenase from Pseudomonas cepacia MBA4 was suggested to serve as a catalytic base [Biochem. J. (1993) 292, 69-74]. In this study, we substituted Asn or Leu for His19 of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which corresponds to His20 of the P. cepacia enzyme. Although the substrate specificity was affected by the substitution, the susceptibilities of substrate halo acids were not substantially diminished, and the Km and kcat values of the mutant enzymes for L-2-chloropropionate were not significantly different from those of the wild-type enzyme. In addition, the wild-type and mutant enzymes showed the same pH optimum. Accordingly, His19 is not essential for catalysis of L-2-halo acid dehalogenase.

Thermostable ornithine aminotransferase from Bacillus sp. YM-2: purification and characterization.

Thermostable L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine: 2-oxo-acid 5-aminotransferase) [EC 2.6.1.13] was purified to homogeneity from Bacillus sp. YM-2. The enzyme has a molecular weight of about 82,000 and consists of two subunits with identical molecular weights. The enzyme catalyzes transamination from L-ornithine to alpha-ketoglutarate, producing L-glutamate and L-glutamate gamma-semialdehyde, which is spontaneously dehydrated to L-delta 1-pyrroline-5-carboxylate, and the enzyme is most active at 70 degrees C. In addition to L-ornithine, the enzyme unexpectedly acts on D-ornithine, the reaction rate being 6% of that for L-ornithine. The enzyme contains 1 mol each of pyridoxal 5'-phosphate and another vitamin B6 compound per mol. The enzyme released the bound pyridoxal 5'-phosphate, as judged from the absorption at 425 nm on incubation with 2.0 M guanidine hydrochloride. The resultant inactive enzyme still gave a 340-nm peak and contained 1 mol of the vitamin B6 compound. The partial amino acid sequence shows high homology with those of mammalian and yeast ornithine delta-aminotransferases.

Thermostable phenylalanine dehydrogenase of Thermoactinomyces intermedius: cloning, expression, and sequencing of its gene.

The gene encoding the thermostable phenylalanine dehydrogenase [EC 1.4.1.-] of a thermophile, Thermoactinomyces intermedius, was cloned and its complete DNA sequence was determined. The phenylalanine dehydrogenase gene (pdh) consists of 1,098 nucleotides and encodes 366 amino acid residues corresponding to the subunit (Mr 41,000) of the hexameric enzyme. The amino acid sequence deduced from the nucleotide sequence of the pdh gene of T. intermedius was 56.0 and 42.1% homologous to those of the phenylalanine dehydrogenases of Bacillus sphaericus and Sporosarcina ureae, respectively. It shows 47.5% homology to that of the thermostable leucine dehydrogenase from B. stearothermophilus. The pdh gene was highly expressed in E. coli JM109, the amount of phenylalanine dehydrogenase produced amounting up to about 8.3% of that of the total soluble protein. We purified the enzyme to homogeneity from transformant cells in a day, with a 58% recovery.

Thermostable alanine racemase of Bacillus stearothermophilus: subunit dissociation and unfolding.

The guanidine hydrochloride-induced subunit dissociation and unfolding of thermostable alanine racemase from Bacillus stearothermophilus have been studied by circular dichroism, fluorescence and absorption spectroscopies, and gel filtration. The overall process was found to be reversible: more than 75% of the original activity was recovered upon reduction of the denaturant concentration. In the range of 0.6 to 1.5 M guanidine hydrochloride, the dimeric enzyme was dissociated into a monomeric form, which was catalytically inactive. The monomeric enzyme appeared to bind the cofactor pyridoxal phosphate by a non-covalent linkage, although the native dimeric enzyme binds the cofactor through an aldimine Schiff base linkage. The monomer was mostly unfolded, with the transition occurring in the range of 1.8 to 2.2 M guanidine hydrochloride.

Enzymatic in situ analysis by 1H-NMR of the hydrogen transfer stereospecificity of NAD(P)+-dependent dehydrogenases.

We have established a simple procedure for the in situ analysis of stereospecificity of an NAD(P)-dependent dehydrogenase for C-4 hydrogen transfer of NAD(P)H by means of glutamate racemase [EC 5.1.13] and glutamate dehydrogenase [EC 1.4.1.3]. Glutamate racemase inherently catalyzes the exchange of alpha-H of glutamate with 2H during racemization in 2H2O. When the reactions of glutamate racemase and glutamate dehydrogenase, which is pro-S specific for the C4-H transfer of NAD(P)H, are coupled in 2H2O, [4S-2H]-NAD(P)H is exclusively produced. Therefore, if 1H is fully retained at C-4 of NAD(P)+ after incubation of a reaction mixture containing both the enzymes and a dehydrogenase to be tested, the stereospecificity of the dehydrogenase is the same as that of glutamate dehydrogenase. When the C4-H of NAD(P)+ is exchanged with 2H, the enzyme to be examined is different from glutamate dehydrogenase in stereospecificity. Thus, we can readily determine the stereospecificity by 1H-NMR measurement of NAD(P)+ without isolation of the coenzymes and products.

Reaction mechanism of glutamate racemase, a pyridoxal phosphate-independent amino acid racemase.

Glutamate racemase of Pediococcus pentosaceus contained no cofactor, and was completely inactivated by a thiol reagent. The role of a cysteine residue in the enzyme reaction was studied by chemical modification. The modification of this cysteine residue resulted in a concomitant loss of activity. DL-Glutamate protected the enzyme from inactivation. The inactivated enzyme was reactivated by addition of dithiothreitol. The racemization in 2H2O showed an overshoot in the optical rotation of glutamate before the substrate was completely racemized. This indicates that the removal of alpha-hydrogen is the rate determining step. During the racemization of D- or L-glutamate in 3H2O, tritium was incorporated preferentially into the product. Glutamate is racemized by the enzyme probably through a two base mechanism.

Chemical synthesis and expression of copper metallothionein gene of Neurospora crassa.

The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene. The MT-beta-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the beta-galactosidase through methionine residues. An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation. The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs.

Cloning and expression of the glutamate racemase gene of Bacillus pumilus.

A glutamate racemase gene (murI) was found in Bacillus pumilus cells and cloned into Escherichia coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B. pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG. The deduced amino acid sequence shows significant similarity with those of glutamate racemases from E. coli (ratio of identical residues, 28%), Pediococcus pentosaceus (44%), and Staphylococcus haemolyticus (49%). B. pumilus MurI was expressed as a fusion protein connected to the N-terminal 12 residues of beta-galactosidase; the fusion protein showed glutamate racemase activity, and resembled the enzyme of P. pentosaceus in physicochemical and enzymological properties.

Construction and properties of a fragmentary D-amino acid aminotransferase.

D-Amino acid aminotransferase [EC 2.6.1.21] catalyzes the inter-conversion between various D-amino acids and alpha-keto acids. The subunit of the homodimeric enzyme from Bacillus sp. YM-1 consists of two domains connected by a single loop, which has no direct contact with the active site residues or the cofactor, pyridoxal 5'-phosphate [Sugio, S., Petsko, G.A., Manning, J.M., Soda, K., and Ringe, D. (1995) Biochemistry 34, 9661-9669]. We constructed two plasmids, one encoding a polypeptide fragment corresponding to the N-terminal domain, and the other a fragment corresponding to the C-terminal domain. When both polypeptide fragments were expressed together in the same host cell, an active fragmentary enzyme consisting of two sets of the two polypeptide fragments was produced. When the two polypeptide fragments were expressed separately, each of them provided a soluble protein but with no activity. However, D-amino acid aminotransferase activity appeared upon incubation of a mixture of the two fragments. The active fragmentary enzyme was purified to homogeneity and characterized; it was found to be similar to the wild-type enzyme in various enzymological properties except substrate specificity, inhibition by alpha-ketoglutarate, and thermostability. The fragmentary enzyme showed higher catalytic activity toward several substrates, such as D-lysine and D-arginine, than the wild-type enzyme.

Transamination as a side-reaction catalyzed by alanine racemase of Bacillus stearothermophilus.

The pyridoxal form of alanine racemase of Bacillus stearothermophilus was converted to the pyridoxamine form by incubation with its natural substrate, D- or L-alanine, under acidic conditions: the enzyme loses its racemase activity concomitantly. The pyridoxamine form of the enzyme returned to the pyridoxal form by incubation with pyruvate at alkaline pH. Thus, alanine racemase catalyzes transamination as a side function. In fact, the apo-form of the enzyme abstracted tritium from [4'-3H]pyridoxamine in the presence of pyruvate. A mutant enzyme containing alanine substituted for Lys39, whose epsilon-amino group forms a Schiff base with the C4' aldehyde of pyridoxal 5'-phosphate in the wild-type enzyme, was inactive as a catalyst for racemization as well as transamination. However, when methylamine was added to the mutant enzyme, it became active in both reactions. These results suggest that the epsilon-amino group of Lys39 participates in both racemization and transamination when catalyzed by the wild-type enzyme.

X-ray structure of a reaction intermediate of L-2-haloacid dehalogenase with L-2-chloropropionamide.

The crystal structure of a complex prepared by soaking a single crystal of the S175A mutant of L-2-haloacid dehalogenase from Pseudomonas sp. YL in a solution containing a poor substrate, L-2-chloropropionamide, has been determined by X-ray analysis at 2. 15 A resolution with a crystallographic R factor of 19.8%. The present analysis has revealed the structure of the reaction intermediate trapped in the crystal. In the intermediate, the substrate moiety lacking chlorine is covalently bound to the carboxyl group of D10, and adopts the pro-D-configuration at the C2 atom. The amide group of the substrate is hydrogen-bonded with the hydroxy group of S118. The methyl group bound to the C2 atom exists in a hydrophobic pocket which is important for recognition of the alkyl group of the substrate. The guanidino group of R41 has reasonable orientation for halogen-abstraction.

Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification.

The gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp. YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues. The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(beta-aminoethyl)cysteine (SAEC), S-(beta-aminopropyl)cysteine (SAPC), and S-(beta-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively. The modified mutant enzymes showed absorbance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0. The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3. This suggests that omega-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate. The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 10(6)-10(8) times more active than the K239C mutant enzyme. These results suggest that omega-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.