Self-protection mechanism in D-cycloserine-producing Streptomyces lavendulae. Gene cloning, characterization, and kinetics of its alanine racemase and D-alanyl-D-alanine ligase, which are target enzymes of D-cycloserine.
An antibiotic, D-cycloserine (DCS), inhibits the catalytic activities of alanine racemase (ALR) and d-alanyl-d-alanine ligase (DDL), which are necessary for the biosynthesis of the bacterial cell wall. In this study, we cloned both genes encoding ALR and DDL, designated alrS and ddlS, respectively, from DCS-producing Streptomyces lavendulae ATCC25233. Each gene product was purified to homogeneity and characterized. Escherichia coli, transformed with a pET vector carrying alrS or ddlS, displays higher resistance to DCS than the same host carrying the E. coli ALR- or DDL-encoded gene inserted into the pET vector. Although the S. lavendulae DDL was competitively inhibited by DCS, the K(i) value (920 microM) was obviously higher (40 approximately 100-fold) than those for E. coli DdlA (9 microM) or DdlB (27 microM). The high K(i) value of the S. lavendulae DDL suggests that the enzyme may be a self-resistance determinant in the DCS-producing microorganism. Kinetic studies for the S. lavendulae ALR suggest that the time-dependent inactivation rate of the enzyme by DCS is absolutely slower than that of the E. coli ALR. We conclude that ALR from DCS-producing S. lavendulae is also one of the self-resistance determinants.
Noda
Masafumi
M
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-Ku, Hiroshima 734-8551, Japan.
Kawahara
Yumi
Y
Ichikawa
Azusa
A
Matoba
Yasuyuki
Y
Matsuo
Hiroaki
H
Lee
Dong-Geun
DG
Kumagai
Takanori
T
Sugiyama
Masanori
M
D000411:Alanine Racemase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D003001:Cloning, Molecular; D003523:Cycloserine / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000378:metabolism*; D013302:Streptomyces / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D003523:Cycloserine; D000411:Alanine Racemase; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase