Correlation between catalytic activity and monomer-dimer equilibrium of bacterial alanine racemases.

From the reaction mechanism and crystal structure analysis, a bacterial alanine racemase is believed to work as a homodimer with a substrate, l-alanine or d-alanine. We analysed oligomerization states of seven alanine racemases, biosynthetic and catabolic, from Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, P. putida and P. fluorescens, with three different methods, gel filtration chromatography, native PAGE and analytical ultracentrifugation. All alanine racemases were proved to be in a dynamic equilibrium between monomeric and dimeric form with every methods used in this study. In both biosynthetic and catabolic alanine racemases, association constants for dimerization were high for the enzymes with high V(max) values. The enzymes with low V(max) values gave the low association constants. We proposed that alanine racemases are classified into two types; the enzymes with low and high-equilibrium association constants for dimerization.

Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15.

The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD(+)-dependent oxidation of the ß-hydroxyl group of l-ß-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO(2).

Methyladeninylcobamide functions as the cofactor of methionine synthase in a Cyanobacterium, Spirulina platensis NIES-39.

To clarify the physiological function of pseudovitamin B(12) (or adeninylcobamide; AdeCba) in Spirulina platensis NIES-39, cobalamin-dependent methionine synthase (MS) was characterized. We cloned the full-length Spirulina MS. The clone contained an open reading frame encoding a protein of 1183 amino acids with a molecular mass of 132 kDa. Deduced amino acid sequences of the Spirulina MS contained critical residues identical to cobalamin-, zinc-, S-adenosylmethionine-, and homocysteine-binding motifs. The recombinant Spirulina enzyme showed higher affinity for methyladeninylcobamide than methylcobalamin as a cofactor. These results indicate that Spirulina cells can utilize AdeCba synthesized as the cofactor for MS.

Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z.

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4 degrees C with 10 microM 5'-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa +/- 5.0 kDa using Superdex 200 gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa +/- 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5'-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.

Molecular characterization of lysine 6-dehydrogenase from Achromobacter denitrificans.

An inducible lysine 6-dehydrogenase (Lys 6-DH), which catalyzes the oxidative deamination of the 6-amino group of L-lysine in the presence of NAD(+), was purified to homogeneity from Achromobacter denitrificans, yielding a homodimeric protein of 80 kDa. The enzyme was specific for the substrate L-lysine and NAD(+) served as a cofactor. The dimeric enzyme associated into a hexamer in the presence of 10 mM L-lysine. The K(m) values for L-lysine and NAD(+) were 5.0 and 0.09 mM, respectively. The lys 6-dh gene was cloned and overexpressed in E. coli. The open reading frame was 1,107 nucleotides long and encoded a peptide containing 368 amino acids with 39,355 Da. The recombinant enzyme was purified to homogeneity and characterized. Enzyme activities and kinetic properties of the recombinant enzyme were almost the same as those of the endogenous enzyme obtained from A. denitrificans. Crystals of the enzyme were obtained using the hanging drop method.

Genetic design of conditional D-glutamate auxotrophy for Bacillus subtilis: use of a vector-borne poly-gamma-glutamate synthetic system.

Bacillus subtilis possesses two glutamate racemase isozymes, RacE and YrpC. For the first time, we succeeded in constructing glutamate racemase-gene disruptants of B. subtilis. Phenotypic analysis of their D-glutamate auxotrophy indicated that the RacE-type glutamate racemase is important for ensuring maximum growth rate but dispensable. The YrpC-type glutamate racemase probably operates as an anaplerotic enzyme for RacE, especially under liquid culture conditions. We found novel applicability of RacE-less mutants inheriting only a marginal activity for endogenous D-glutamate supply, viz. the employment for the in vivo identification of D-glutamate-consuming systems. In fact, the genetic induction of a poly-gamma-glutamate synthetic system led a RacE-less mutant to severe growth suppression, which was overcome in the presence of a high concentration of exogenous D-glutamate. The results indicate that a significant amount of D-glutamate is consumed during poly-glutamate biosynthesis. To our knowledge, this is the first report of conditional D-glutamate auxotrophy for B. subtilis.

Novel poly-gamma-glutamate-processing enzyme catalyzing gamma-glutamyl DD-amidohydrolysis.

The pgdS gene product of Bacillus subtilis, PgdS, cleaves poly-gamma-glutamate (PGA) in an endo-peptidase-like fashion. However, its catalytic property remains obscure. In this study, a simple assay for the PgdS enzyme using 1-fluoro-2,4-dinitrobenzene was developed, and some characteristics of PgdS, such as optimal pH, were examined. The enzyme was strongly inhibited by a thiol-modifying reagent, suggesting that it possesses essential cysteine residue(s) in catalysis. PgdS exhibited a high affinity to PGA that consisted mainly of D-glutamate residues, but no affinity to PGA composed only of L-glutamate residues (L-PGA). The enzyme processed DL-copolymer-type PGA (DL-PGA) with an average molecular mass of 1,000 kDa to a high-molecular-mass L-glutamate-rich fragment (average 200 kDa), the L-rich PGA fragment, and low-molecular-mass fragment composed mostly of D-glutamate residues (average 5 kDa), D-fragment. To deepen our understanding of the catalytic property of the PgdS enzyme, we analyzed the structures of the N- and C-terminal regions and found that D-glutamyl residues successively lie even at both ends of the L-rich PGA fragment. Our observations indicate that PgdS is a novel endo-peptidase that specifically cleaves the gamma-amide linkage between two D-glutamate residues in PGA, i.e., gamma-glutamyl DD-amidohydrolase. The enzyme is possibly useful in the biochemical processing of B. subtilis DL-PGA.

Genetically engineered poly-gamma-glutamate producer from Bacillus subtilis ISW1214.

The pgsBCA-gene disruptant from Bacillus subtilis ISW1214, i.e., MA41, does not produce poly-gamma-glutamate (PGA). We newly constructed an MA41 recombinant bearing the plasmid-borne PGA synthetic system, in which PGA production was strictly controlled by the use of xylose. Unlike the parent strain, ISW1214, the genetically engineered strain produced abundant PGA in both L-glutamate-rich and D-glutamate-rich media.

Directed evolution of extradiol dioxygenase by a novel in vivo DNA shuffling.

RecA-dependent homologous recombination in Escherichia coli is a very effective way to construct chimeras between two homologous genes. The disadvantage of in vivo method is a small library size of chimeric genes in comparison with in vitro DNA shuffling. In order to overcome the disadvantage, we have developed novel in vivo DNA shuffling methods with successive homologous recombinations. Linearized DNA molecules with two homologous genes were made with ligation rather than the conventional restriction enzyme cleavage between two genes. The three-way ligation of a vector and two homologous bphC genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenases or the two-way ligation of the donor bphC gene and an acceptor plasmid carrying the homologous bphC gene generated a variety of linearized DNA molecules. The homologous recombination between the genes on the linearized DNA molecules created the large chimeric bphC gene libraries in a recBC sbcA E. coli strain. After three rounds of recombinations, chimeric bphC genes with four-part gene fragments by triple-crossover were easily obtained. By employing a 96-well microtiter plate high-throughput screening, thermally stable chimeric 2,3-dihydroxybiphenyl 1,2-dioxygenases were selected from chimeric bphC gene libraries. This opens up a new way for directed evolution of proteins in vivo.

Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli.

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.

Directed evolution of bacterial alanine racemases with higher expression level.

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the interconversion of L-alanine and D-alanine. It can be classified into two groups: biosynthetic enzymes with low catalytic activity and catabolic enzymes with high catalytic activity. It can react with serine to a limited extent. Two biosynthetic alanine racemase genes in Escherichia coli and Salmonella typhimurium were DNA shuffled, and a very diverse chimeric gene library was constructed. An E. coli serine auxotroph was transformed with the shuffled genes, and the recombinant clones were screened on selective media supplemented with 0.5-5 mM D-serine as an L-serine supplier. Selected clones were expected to contain racemases exhibiting higher catalytic activities toward alanine as well as serine. Three independent clones that grew on selective media were isolated. The specific activities of crude extracts prepared from cells expressing the chimeric racemases were increased up to approximately three times more than those expressing the parental enzymes. The best chimera Ser15 racemase was expressed at a level approximately twofold higher than the parental alanine racemases. This high protein expression was demonstrated to be posttranscriptionally achieved.

Characterization of an inducible phenylserine aldolase from Pseudomonas putida 24-1.

An inducible phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of L-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5' phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on L-threo-3-phenylserine (Km, 1.3 mM), l-erythro-3-phenylserine (Km, 4.6 mM), l-threonine (Km, 29 mM), and L-allo-threonine (Km, 22 mM). In the reverse reaction, threo- and erythro- forms of L-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from Pseudomonas putida 24-1 was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5'-phosphate.

Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum.

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains d-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understand D-aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of D- and L-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially the D-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of the B. bifidum enzyme.

Enzymatic synthesis of high-molecular-mass poly-gamma-glutamate and regulation of its stereochemistry.

For the first time, we succeeded in synthesizing in vitro poly-gamma-glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp. chungkookjang. The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles. The optimum pH for the reaction was found to be approximately 7.0. We examined the effects of some divalent cations on PGA synthesis and found that Mg(2+) was essential in catalysis and that Zn(2+) additionally boosted the activity. In contrast, Fe(2+) and Ca(2+) acted as inhibitors. Mn(2+) did not apparently influence the in vitro formation of PGA. DL-Glutamate (D isomer content, 60 to 80%) apparently served as the best substrate; d-Glutamate was preferable to the L isomer as a substrate. When D- and L-glutamate were used for the reaction, the elongated chains of PGAs were composed of the D- and L-isomers, respectively. Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (DL ratio) of glutamate as the substrate. Furthermore, genetic analysis indicated that all the pgsB, -C, and -A gene products, which are responsible for PGA production by B. subtilis cells, were also indispensable for enzymatic PGA synthesis.

Molecular breeding of 2,3-dihydroxybiphenyl 1,2-dioxygenase for enhanced resistance to 3-chlorocatechol.

3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.

Differences in effects on DNA gyrase activity between two glutamate racemases of Bacillus subtilis, the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC (MurI) isozyme.

Bacillus subtilis possesses two isogenes encoding glutamate racemases, the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC isozyme, and produces abundant amounts of the Glr enzyme. The YrpC isozyme, but not the Glr enzyme, was found to influence the activity of DNA gyrase, as did the MurI-type glutamate racemase of Escherichia coli, which is involved in peptidoglycan synthesis during cell division.

Cloning and overexpression of the 3-hydroxyisobutyrate dehydrogenase gene from pseudomonas putida E23.

The structural gene for NAD+-dependent 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from Pseudomonas putida E23 was cloned in Escherichia coli cells to obtain a large amount of the enzyme and its nucleotides were sequenced to study its structural relationship with other proteins. The gene encoded a polypeptide containing 295 amino acid residues and was in a cluster with the gene for methylmalonate semialdehyde dehydrogenase. Transformed E. coli cells overproduced 3-hydroxyisobutyrate dehydrogenase, and the recombinant enzyme was purified to homogeneity with a high yield. Lysine and asparagine residues, which are important in catalysis of the 3-hydroxyacid dehydrogenase family, are conserved in this enzyme.

Characterization of short-chain dehydrogenase/reductase homologues of Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C).

Short-chain dehydrogenase/reductase homologues from Escherichia coli (YdfG) and Saccharomyces cerevisiae (YMR226C) show high sequence similarity to serine dehydrogenase from Agrobacterium tumefaciens. We cloned each gene encoding YdfG and YMR226C into E. coli JM109 and purified them to homogeneity from the E. coli clones. YdfG and YMR226C consist of four identical subunits with a molecular mass of 27 and 29 kDa, respectively. Both enzymes require NADP(+) as a coenzyme and use L-serine as a substrate. Both enzymes show maximum activity at about pH 8.5 for the oxidation of L-serine. They also catalyze the oxidation of D-serine, L-allo-threonine, D-threonine, 3-hydroxyisobutyrate, and 3-hydroxybutyrate. The k(cat)/K(m) values of YdfG for L-serine, D-serine, L-allo-threonine, D-threonine, L-3-hydroxyisobutyrate, and D-3-hydroxyisobutyrate are 105, 29, 199, 109, 67, and 62 M(-1) s(-1), and those of YMR226C are 116, 110, 14600, 7540, 558, and 151 M(-1) s(-1), respectively. Thus, YdfG and YMR226C are NADP(+)-dependent dehydrogenases acting on 3-hydroxy acids with a three- or four-carbon chain, and L-allo-threonine is the best substrate for both enzymes.

Glutamate racemase is an endogenous DNA gyrase inhibitor.

Almost all bacteria possess glutamate racemase to synthesize d-glutamate as an essential component of peptidoglycans in the cell walls. The enforced production of glutamate racemase, however, resulted in suppression of cell proliferation. In the Escherichia coli JM109/pGR3 clone, the overproducer of glutamate racemase, the copy number (i.e. replication efficiency) of plasmid DNA declined dramatically, whereas the E. coli WM335 mutant that is defective in the gene of glutamate racemase showed little genetic competency. The comparatively low and high activities for DNA supercoiling were contained in the E. coli JM109/pGR3 and WM335 cells, respectively. Furthermore, we found that the DNA gyrase of E. coli was modulated by the glutamate racemase of E. coli in the presence of UDP-N-acetylmuramyl-l-alanine, which is a peptidoglycan precursor and functions as an absolute activator for the racemase. This is the first finding of the enzyme protein participating in both d-amino acid metabolism and DNA processing.

Cloning and sequencing of the serine dehydrogenase gene from Agrobacterium tumefaciens.

The structural gene for NADP+-dependent serine dehydrogenase [EC 1.1.1.-] from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed. The gene encodes a polypeptide containing 249 amino acid residues. The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae.