Self-protection Mechanism in d-Cycloserine-producing Streptomyces lavendulae

Abstract

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 M) was obviously higher (40ϳ100-fold) than those for E. coli DdlA (9 M) or DdlB (27 M). 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.

Structural Evidence That Alanine Racemase from a d-Cycloserine-producing Microorganism Exhibits Resistance to Its Own Product

“…This observation means that the conversion of DCS to a final form, which noncompetitively inhibits the enzymatic activity, is significantly slow in Streptomyces ALR, resulting in the resistance to DCS (34). In the crystal structure of the DCS-bound form of the Streptomyces ALR, the C␣ atom of the final DCS-derivative in site A is located near the hydroxyl of Tyr 270 Ј, whereas that in site B is near the amino group of Lys 38 .…”

“…Crystallography of Streptomyces ALR-Enzyme expression and purification of the Streptomyces ALR were done as described previously (34). Prior to crystallization, the purified enzyme solution was dialyzed against a 30 mM Tris-HCl buffer (pH 8.5) containing 50 mM NaCl and 0.5 mM PLP and then concentrated to 10 mg/ml using an Amicon concentrator (Millipore).…”

“…In addition, we performed kinetic studies for enzyme inhibition by DCS and LCS on Streptomyces ALR, which suggest that the time-dependent inhibitory rate of DCS is faster than that of LCS. Interestingly, the inhibitory rate of the cycloserine enantiomers to Streptomyces ALR was about 3-fold lower than that of the E. coli ALR (accompanying article (34)) and 4-fold lower than that of B. stearothermophilus ALR (13), both of which are susceptible to DCS. This result must be distinguished from a DCS resistance mechanism proposed in Mycobacterium, in which overexpression of ALR contributes to DCS resistance (21,22).…”

“…We noticed that both ALR and D-Ala-D-Ala ligase, targets of DCS from a DCS producer, exhibit resistance to DCS. Therefore, we have cloned genes encoding these two enzymes from DCS-producing S. lavendulae ATCC25233 and characterized their gene products (accompanying article (34)). In addition, we performed kinetic studies for enzyme inhibition by DCS and LCS on Streptomyces ALR, which suggest that the time-dependent inhibitory rate of DCS is faster than that of LCS.…”

High-Level Heterologous Production of d -Cycloserine by Escherichia coli

“…The functions of dcsI and dcsJ had previously been analyzed using the corresponding genes cloned from other D-CS-producing strains, i.e., S. lavendulae ATCC 25233 (11) and S. garyphalus (CSH) 5-12 (12), demonstrating that both gene products are responsible for self-resistance in the D-CS producer. Gene disruption and recombinant protein analyses have demonstrated that the revised D-CS biosynthetic pathway is as follows.…”