S. Kato et al.
and serine but did not display any lysine racemization
no significant effects on the kinetic parameters towards
alanine (Table I). The double mutant, I222T/Y354W,
reacted with almost all the standard amino acids
2. Kato, S., Ishihara, T., Hemmi, H., Kobayashi, H., and
Yoshimura, T. (2011) Alterations in D-amino acid con-
centrations and microbial community structures during
the fermentation of red and white wines. J. Biosci.
Bioeng. 111, 104108
3. Inagaki, K., Tanizawa, K., Badet, B., Walsh, C.T.,
Tanaka, H., and Soda, K. (1986) Thermostable alanine
racemase from Bacillus stearothermophilus: molecular
cloning of the gene, enzyme purification, and character-
ization. Biochemistry 25, 32683274
Lysine racemization activity was also enhanced, and
value towards lysine of the I222T/
ꢁ1
the kcatꢀKm
Y354W alanine racemase mutant was comparable
with that of the wild-type lysine racemase (Table I).
Considering that only the double mutant exhibited ac-
tivity towards lysine, these mutations might synergis-
tically affect substrate specificity and, in particular, the
specificity for lysine. The double mutant still retained
4. Soda, K. and Osumi, T. (1969) Crystalline amino acid
racemase with low substrate specificity. Biochem.
Biophys. Res. Comm. 35, 363368
5. Matsui, D., Oikawa, T., Arakawa, N., Osumi, S.,
Lausberg, F., Stabler, N., Freudl, R., and Eggeling, L.
¨
ꢁ1
ꢃ50% of the wild-type kcatꢀKm values towards D-
(2009) A periplasmic, pyridoxal-50-phosphate-dependent
amino acid racemase in Pseudomonas taetrolens. Appl.
Microbiol. Biotechol. 83, 10451054
and L-alanine (Table I). The mutation studies of ala-
nine racemase led us to the same conclusion as that
obtained through the mutation studies of lysine race-
mase: Thr224 and Trp355 of lysine racemase provide
the structure that is essential for accommodating lysine
as the substrate, but Ile222 and Tyr354, the corres-
ponding residues of alanine racemase, are not critical
for alanine racemization. However, mutation of these
residues altered the substrate specificity of the alanine
racemase. In alanine racemase, the a-amino group of
Ile222 and the hydroxyl group of Tyr354 have been
suggested to interact with the phosphate group of
PLP through a hydrogen bond (12). Mutation of
these residues probably affects the hydrogen bond to
alter the substrate specificity.
6. Tanizawa, K., Masu, Y., Asano, S., Tanaka, H., and
Soda, K. (1989) Thermostable D-amino acid amino-
transferase from
a
thermophilic Bacillus species.
Purification, characterization, and active site sequence
determination. J. Biol. Chem. 264, 24452449
7. Tanizawa, K., Yoshimura, T., and Soda, K. (1985)
L-Lysine transaminase from Flavobacterium lutescens.
Methods Enzymol. 113, 96102
8. Kurokawa, Y., Watanabe, A., Yoshimura, T., Esaki, N.,
and Soda, K. (1998) Transamination as a side-reaction
catalyzed by alanine racemase of Bacillus stearothermo-
philus. J. Biochem. 124, 11631169
9. Briggs, G.E. and Haldane, J.B.S. (1925) A note on the
kinetics of enzyme action. Biochem. J. 19, 338339
10. Walsh, C.T. (1989) Enzymes in the D-alanine branch of
bacterial cell wall peptidoglycan assembly. J. Biol. Chem.
264, 23932396
In this study, we demonstrated that one of the ala-
nine racemase homologs of O. oeni (OEOE0162) is a
lysine racemase, which is highly specific for basic
amino acids. D-Lys acts as a catabolic nutrient in
Pseudomonas taetrolens (5) and is a component of the
cell wall peptidoglycan in Thermotoga maritima (15).
However, the physiological importance of D-Lys in
O. oeni is still unknown. Our future task is to elucidate
the physiological functions of D-Lys and the lysine
racemase OEOE0162 in O. oeni cells.
11. Shaw, J.P., Pestko, G.A., and Ringe, D. (1997)
Determination of the structure of alanine racemase
˚
from Bacillus stearothermophilus at 1.9-A resolution.
Biochemistry 11, 13291342
12. Watanabe, A., Yoshimura, T., Mikami, B., Hayashi, H.,
Kagamiyama, H., and Esaki, N. (2002) Reaction mech-
anism of alanine racemase from Bacillus stearothermo-
philus: x-ray crystallographic studies of the enzyme
bound with N-(50-phosphopyridoxyl)alanine. J. Biol.
Chem. 277, 1916619672
Supplementary Data
13. Neidhart, D.J., Distefano, M.D., Tanizzawa, K., Soda,
K., Walsh, C.T., and Petsko, G.G. (1987) X-ray crystal-
lographic studies of the alanine-specific racemase
from Bacillus stearothermophilus. J. Biol. Chem. 262,
1532315326
14. Yoshimura, T. (2005) Yeast enzymes involved in
D-amino acid metabolism. Vitamins 79, 277283
15. Boniface, A., Parquet, C., Arthur, M., Mengin-Lecreulx,
D., and Blanot, D. (2009) The elucidation of the struc-
ture of Thermotoga maritima peptidoglycan reveals
two novel types of cross-link. J. Biol. Chem. 284,
2185621862
Acknowledgements
This work was supported by the Program for Promotion of Basic
and Applied Researches for Innovation in Bio-oriented Industry
(BRAIN).
Conflict of interest
None declared.
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