Crystallographic studies of aspartate racemase from Lactobacillus sakei NBRC 15893.

Aspartate racemase catalyzes the interconversion between L-aspartate and D-aspartate and belongs to the PLP-independent racemases. The enzyme from the lactic acid bacterium Lactobacillus sakei NBRC 15893, isolated from kimoto, is considered to be involved in D-aspartate synthesis during the brewing process of Japanese sake at low temperatures. The enzyme was crystallized at 293 K by the sitting-drop vapour-diffusion method using 25%(v/v) PEG MME 550, 5%(v/v) 2-propanol. The crystal belonged to space group P3121, with unit-cell parameters a = b = 104.68, c = 97.29 Å, and diffracted to 2.6 Šresolution. Structure determination is under way.

Principal component analysis of the relationship between the D-amino acid concentrations and the taste of the sake.

We performed sensory evaluations on 141 bottles of sake and analyzed the relationship between the D-amino acid concentrations, and the taste of the sake using principal component analysis, which yielded seven principal components (PC1-7) that explained 100 % of the total variance in the data. PC1, which explains 33.6 % of the total variance, correlates most positively with strong taste and most negatively with balanced tastes. PC2, which explains 54.4 % of the total variance, correlates most positively with a sweet taste and most negatively with bitter and sour tastes. Sakes brewed with "Kimoto yeast starter" and "Yamahaimoto" had high scores for PC1 and PC2, and had strong taste in comparison with sakes brewed with "Sokujo-moto". When present at concentrations below 50 µM, D-Ala did not affect the PC1 score, but all the sakes showed a high PC1 score, when the D-Ala was above 100 µM. Similar observations were found for the D-Asp and D-Glu concentrations with regard to PC1, and the threshold concentrations of D-Asp and D-Glu that affected the taste were 33.8 and 33.3 µM, respectively. Certain bacteria present in sake, especially lactic acid bacteria, produce D-Ala, D-Asp and D-Glu during storage, and these D-amino acids increased the PC1 score and produced a strong taste (Nojun). When D- and L-Ala were added to the sakes, the value for the umami taste in the sensory evaluation increased, with the effect of D-Ala being much stronger than that of L-Ala. The addition of 50-5,000 µM DL-Ala did not effect on the aroma of the sakes at all.

High-performance liquid chromatography analysis of naturally occurring D-amino acids in sake.

We measured all of the D- and L-amino acids in 141 bottles of sakes using HPLC. We used two precolumn derivatization methods of amino acid enantiomer detection with o-phthalaldehyde and N-acetyl-L-cysteine, as well as (+)-1-(9-fluorenyl)ethyl chloroformate/1-aminoadamantane and one postcolumn derivatization method with o-phthalaldehyde and N-acetyl-L-cysteine. We found that the sakes contained the D-amino acids forms of Ala, Asn, Asp, Arg, Glu, Gln, His, Ile, Leu, Lys, Ser, Tyr, Val, Phe, and Pro. We were not able to detect D-Met, D-Thr D-Trp in any of the sakes analyzed. The most abundant D-Ala, D-Asp, and D-Glu ranged from 66.9 to 524.3 µM corresponding to relative 34.4, 12.0, and 14.6% D-enantiomer. The basic parameters that generally determine the taste of sake such as the sake meter value (SMV; "Nihonshudo"), acidity ("Sando"), amino acid value ("Aminosando"), alcohol content by volume, and rice species of raw material show no significant relationship to the D-amino acid content of sake. The brewing water ("Shikomimizu") and brewing process had effects on the D-amino acid content of the sakes: the D-amino acid contents of the sakes brewed with deep-sea water "Kaiyoushinosousui", "Kimoto yeast starter", "Yamahaimoto", and the long aging process "Choukijukusei" are high compared with those of other sakes analyzed. Additionally, the D-amino acid content of sakes that were brewed with the adenine auxotroph of sake yeast ("Sekishoku seishu kobo", Saccharomyces cerevisiae) without pasteurization ("Hiire") increased after storage at 25 °C for three months.

Site-directed mutagenesis of rice serine racemase: evidence that Glu219 and Asp225 mediate the effects of Mg2+ on the activity.

We succeeded in constructing the Glu219Ala/Asp225Ala (i.e., E219A/D225A) serine racemase (SerR) by site-directed mutagenesis, and the effects of Mg(2+) on the catalytic efficiency and the structure were compared between the E219A/D225A-SerR and the wild-type protein. This is the first example of a serine racemase whose amino acid residues in the Mg(2+)-binding site were replaced with other amino acids by site-directed mutagenesis. Neither the serine racemase nor the dehydratase activities of the E219A/D225A-SerR were affected by the addition of Mg(2+), and Glu219 and Asp225 of the SerR are the essential amino acid residues for Mg(2+) to affect both kinds of enzyme activities. Therefore, Glu219 and Asp225 mediate the effects of Mg(2+) on the activity and are important for the SerR to form the Mg(2+)-binding site. Judging from the difference of the K(eq) values between the E219A/D225A-SerR and the SerR, Mg(2+) might affect the equilibrium states in the racemase reaction. The fluorescence quenching analysis of the E219A/D225A-SerR showed that Mg(2+) bound to Glu219 and Asp225 of the SerR probably causes a conformational change in the ternary structure of the SerR.

Occurrence of D-serine in rice and characterization of rice serine racemase.

Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D+L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 degrees C and pH9.0) and for the dehydratase reaction (35 degrees C and pH9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg(2+) increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg(2+), and this structural change probably regulates the two enzymatic activities.