Engineering the substrate specificity of Alcaligenes D-aminoacylase useful for the production of D-amino acids by optical resolution.

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (AxD-NAase) offers a novel biotechnological application, the production of D-amino acid from the racemic mixture of N-acyl-DL-amino acids. However, its substrate specificity is biased toward certain N-acyl-D-amino acids. To construct mutant AxD-NAases with substrate specificities different from those of wild-type enzyme, the substrate recognition site of the AxD-NAase was rationally manipulated based on computational structural analysis and comparison of its primary structure with other D-aminoacylases with distinct substrate specificities. Mutations of amino acid residues, Phe191, Leu298, Tyr344, and Met346, which interact with the side chain of the substrate, induced marked changes in activities toward each substrate. For example, the catalytic efficiency (k(cat)/K(m)) of mutant F191W toward N-acetyl-D-Trp and N-acetyl-D-Ala was enhanced by 15.6- and 1.5-folds, respectively, compared with that of the wild-type enzyme, and the catalytic efficiency (k(cat)/K(m)) of mutant L298A toward N-acetyl-D-Trp was enhanced by 4.4-folds compared with that of the wild-type enzyme. Other enzymatic properties of both mutants, such as pH and temperature dependence, were the same as those of the wild-type enzyme. The F191W mutant in particular is considered to be useful for the enzymatic production of D-Trp which is an important building block of some therapeutic drugs.

Analysis of essential amino acid residues for catalytic activity of glutaminase from Micrococcus luteus K-3.

Structural-based mutational analysis of salt-tolerant glutaminase from Micrococcus luteus K-3 (Micrococcus glutaminase) revealed that three amino acid residues, S64, K67, and E160, were essential to a catalytic reaction. The result suggested that Micrococcus glutaminase had a possible catalytic mechanism similar to class A beta-lactamase rather than glutaminase-asparaginase from Pseudomonas 7A.

Crystal structure of a major fragment of the salt-tolerant glutaminase from Micrococcus luteus K-3.

Glutaminase of Micrococcus luteus K-3 (intact glutaminase; 48kDa) is digested to a C-terminally truncated fragment (glutaminase fragment; 42kDa) that shows higher salt tolerance than that of the intact glutaminase. The crystal structure of the glutaminase fragment was determined at 2.4A resolution using multiple-wavelength anomalous dispersion (MAD). The glutaminase fragment is composed of N-terminal and C-terminal domains, and a putative catalytic serine-lysine dyad (S64 and K67) is located in a cleft of the N-terminal domain. Mutations of the S64 or K67 residues abolished the enzyme activity. The N-terminal domain has abundant glutamic acid residues on its surface, which may explain its salt-tolerant mechanism. A diffraction analysis of the intact glutaminase crystals (a twinning fraction of 0.43) located the glutaminase fragment in the unit cell but failed to turn up clear densities for the missing C-terminal portion of the molecule.

Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40 is salt-tolerant.

Aspergillus oryzae RIB40 possesses the gene of glutaminase (Micrococcus luteus K-3-type glutaminase; AoGls), which has 40% homology with the salt-tolerant glutaminase from M. luteus K-3 (Micrococcus glutaminase). It was found that AoGls is a salt-tolerant enzyme, and its properties are similar to those of Micrococcus glutaminase.

A metal ion as a cofactor attenuates substrate inhibition in the enzymatic production of a high concentration of D-glutamate using N-acyl-D-glutamate amidohydrolase.

N-Acyl-D-glutamate amidohydrolase (D-AGase) was inhibited by 94 % when 1 mol/l N-acetyl-DL-glutamate was used as a substrate. The addition of 1 mM Co2+ stabilized D-AGase. Moreover, the substrate inhibition was weakened to 88% with the addition of 0.4 mM Co2+ to the reaction mixture. Although D-AGase is a zinc-metalloenzyme, the addition of Zn2+ from 0.01 to 10 mM did not increase the D-glutamic acid production in the saturated substrate. Under optimal conditions, 0.38 M D-glutamic acid was obtained from N-acyl-DL-glutamate with 100% of the theoretical yield after 48 h.

Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation.

Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA. Besides L: -glutamine, which was hydrolyzed with the highest specific activity (100%), L: -asparagine (74%), D: -glutamine (75%), and D: -asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60 degrees C, respectively. The enzyme was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0-10.0). About 70 and 50% of enzyme activity was retained even after treatment at 60 and 70 degrees C, respectively, for 10 min. The enzyme showed high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher salt tolerance and thermal stability than bacterial glutaminases that have been reported so far. In a model reaction of Japanese soy sauce fermentation, glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with glutaminases from Aspergillus oryzae, Escherichia coli, Pseudomonas citronellolis, and Micrococcus luteus, indicating that this enzyme is suitable for application in Japanese soy sauce fermentation.

Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b.

A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90 degrees C for up to 1 h. Its optimal pH and temperature were observed to be 7.6-7.8 and 60 degrees C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K(m) and V(max) values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 micromol min(-1) mg(-1) protein and 1,149 micromol min(-1) mg(-1) protein, respectively. The turnover rate (k(cat)) and catalytic efficiency (k(cat)/ K(m)) for Leu-p-NA and LeuGlyGly were 10,179 s(-1) and 49,543 s(-1) and 15,470 mM(-1 ) s(-1) and 1981.7 mM(-1 ) s(-1), respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, beta-mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co(2+) .

Role of arginine residues of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6.

To investigate the role of arginine in the folding of d-aminoacylase, seven arginine residues, R26, R152, R296, R302, R354, R377, and R391, among twelve arginine residues highly conserved in d-aminoacylase, N-acyl-d-aspartate amidohydrolase (d-AAase), and N-acyl-d-glutamate amidohydrolase (d-AGase) from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were substituted with lysine by site-directed mutagenesis. The mutants, R26K, R152K, R296K, and R302K were identified as mutations that increase partitioning of the enzyme into inclusion bodies. No mutants with substitutions within the carboxyterminal segment were found to increase partitioning into inclusion bodies (R354K, R377K, and R392K). These results suggest that arginine residues that position between the N-terminus and central region can play an important role in facilitating folding or stabilizing the structure of d-aminoacylase. By anaerobic cultivation, the production level of R302K in the soluble fraction was improved. Coexpression of the DnaK-DnaJ-GrpE chaperone assisted the folding of R302K, and reduced the effect of the aeration conditions on the solubility of R302K. We hypothesized that R302K requires a larger amount of chaperones for efficient folding than the wild type enzyme.

Site-directed mutagenesis alters DnaK-dependent folding process.

The overproduction of d-aminoacylase (A6-d-ANase) of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) is accompanied by aggregation of the overproduced protein, and its soluble expression is facilitated by the coexpression of DnaK-DnaJ-GrpE (DnaKJE). When the A6-d-ANase gene was expressed in the Escherichia coli dnaK mutant dnaK756, little activity was observed in the soluble fraction, and it was restored by the coexpression of DnaKJE or the substitution of the R354 residue of A6-d-ANase for lysine. These results suggest that the guanidino group of the R354 residue of A6-d-ANase disturbs its proper folding in the absence of DnaK and the disturbance is eliminated by binding of DnaK to the R354 residue in the presence of DnaK. This is the first report that the DnaK-dependent folding process of the enzyme is altered by site-directed mutagenesis.

Molecular cloning, overexpression, and purification of Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40.

We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase). AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli. The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S. cerevisiae and a soluble protein in E. coli. The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E. coli Rosetta (DE3). AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL. This is the first report on the overexpression and purification of a M. luteus K-3-type glutaminase cloned from an eucaryote.

Molecular chaperones facilitate the soluble expression of N-acyl-D-amino acid amidohydrolases in Escherichia coli.

The overproduction of D-aminoacylase ( D-ANase, 233.8 U/mg), N-acyl-D-glutamate amidohydrolase (D-AGase, 38.1 U/mg) or N-acyl-D-aspartate amidohydrolase (D-AAase, 6.2 U/mg) in Escherichia coli is accompanied by aggregation of the overproduced protein. To facilitate the expression of active enzymes, the molecular chaperones GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), trigger factor (TF), GroELS and DnaKJE or GroELS and TF were coexpressed with the enzymes. D-ANase (313.3 U/mg) and D-AGase (95.8 U/mg) were overproduced in an active form at levels 1.3- and 1.8-fold higher, respectively, upon co-expression of GroELS and TF. An E. coli strain expressing the D-AAase gene simultaneously with the TF gene exhibited a 4.3-fold enhancement in d-AAase activity (32.0 U/mg) compared with control E. coli expressing the D-AAase gene alone.

Digestion by serine proteases enhances salt tolerance of glutaminase in the marine bacterium Micrococcus luteus K-3.

Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment. The N-terminus of the glutaminase fragment was the same as that of intact glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior. The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on glutaminase.

Unique amino acid composition of proteins in halophilic bacteria.

The amino acid compositions of proteins from halophilic archaea were compared with those from non-halophilic mesophiles and thermophiles, in terms of the protein surface and interior, on a genome-wide scale. As we previously reported for proteins from thermophiles, a biased amino acid composition also exists in halophiles, in which an abundance of acidic residues was found on the protein surface as compared to the interior. This general feature did not seem to depend on the individual protein structures, but was applicable to all proteins encoded within the entire genome. Unique protein surface compositions are common in both halophiles and thermophiles. Statistical tests have shown that significant surface compositional differences exist among halophiles, non-halophiles, and thermophiles, while the interior composition within each of the three types of organisms does not significantly differ. Although thermophilic proteins have an almost equal abundance of both acidic and basic residues, a large excess of acidic residues in halophilic proteins seems to be compensated by fewer basic residues. Aspartic acid, lysine, asparagine, alanine, and threonine significantly contributed to the compositional differences of halophiles from meso- and thermophiles. Among them, however, only aspartic acid deviated largely from the expected amount estimated from the dinucleotide composition of the genomic DNA sequence of the halophile, which has an extremely high G+C content (68%). Thus, the other residues with large deviations (Lys, Ala, etc.) from their non-halophilic frequencies could have arisen merely as "dragging effects" caused by the compositional shift of the DNA, which would have changed to increase principally the fraction of aspartic acid alone.

Crystallization and preliminary X-ray crystallographic studies of salt-tolerant glutaminase from Micrococcus luteus K-3.

Glutaminase from the marine bacterium Micrococcus luteus K-3 (Micrococcus glutaminase) is a salt-tolerant protein which shows equivalent activities both in the absence and the presence of 3 M sodium chloride and is distinct from halophilic proteins, which are inactivated in the absence of salt. To investigate the mechanisms of the salt-tolerant adaptation of Micrococcus glutaminase, the glutaminase and its major fragment containing about 80% of the protein were crystallized using the hanging-drop vapour-diffusion method. The glutaminase crystals belong to space group P622, with unit-cell parameters a = b = 111.4, c = 210.9 A, alpha = beta = 90, gamma = 120 degrees, and diffract to 2.6 A resolution. The fragment crystals belong to space group F222, with unit-cell parameters a = 115.7, b = 116.4, c = 144.9 A, alpha = beta = gamma = 90 degrees, and diffract to 2.4 A resolution. Data from selenomethionine (SeMet) substituted glutaminase crystals and from SeMet-substituted fragment crystals were collected to 2.6 and 2.4 A resolution, respectively. Structural analyses of the glutaminase and its fragment are currently being attempted using the multiwavelength anomalous diffraction (MAD) phasing method.