Structural evidence that alanine racemase from a D-cycloserine-producing microorganism exhibits resistance to its own product
Structural evidence that alanine racemase from a D-cycloserine-producing microorganism exhibits resistance to its own product
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DOI：
10.1074/jbc.m404605200
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发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Sugiyama, M
中科院分区：
文献类型：
--
作者：
Noda, M;Matoba, Y;Sugiyama, M
丙氨酸消旋酶(ALR)是一种催化丙氨酸对映体相互转化的酶，对细菌细胞壁的合成是必不可少的。结果表明，D-环丝氨酸链霉菌产D-环丝氨酸链霉菌ALR的催化活性比产D-环丝氨酸链霉菌的大肠杆菌ALR更难抑制。为了获得链霉菌ALR对DCs具有抗性这一事实的结构证据，我们在高分辨率下确定了链霉菌ALR的环丝氨酸无对映体和环丝氨酸结合对映体的X射线晶体结构的精确性质。链霉菌ALR具有二聚体结构，它是由一个单体的N-末端结构域与其伴侣的C-末端结构域相互作用形成的。由于二聚体结构的形成，ALR的两个活性部位都由吡哆醛5‘-磷酸(PLP)、PLP结合残基Lys(38)和吡哆醛辅因子的直接环境中的氨基酸组成。目前的模型表明，链霉菌ALR的每个活性部位都保持着比先前确定的脂肪嗜热芽孢杆菌ALR更大的空间和更刚性的构象。此外，我们还发现，链霉菌ALR会缓慢地转化为最终形式的吡哆醛衍生物，这是由环丝氨酸的任一异构体产生的，这抑制了催化活性。事实上，与PLP结合的每个酶结合的环丝氨酸衍生物都具有不对称结构，这证实了缓慢的转化。
Alanine racemase (ALR), an enzyme that catalyzes the interconversion of Ala enantiomers, is essential for the synthesis of the bacterial cell wall. We have shown that it is harder to inhibit the catalytic activity of ALR from D-cycloserine (DCS)-producing Streptomyces lavendulae than that from Escherichia coli by DCS. To obtain structural evidence for the fact that Streptomyces ALR displays resistance to DCS, we determined the precise nature of the x-ray crystal structures of the cycloserine-free and cycloserine enantiomer-bound forms of Streptomyces ALR at high resolutions. Streptomyces ALR takes a dimer structure, which is formed by interactions between the N-terminal domain of one monomer with the C-terminal domain of its partner. Each of the two active sites of ALR, which is generated as a result of the formation of the dimer structure, is composed of pyridoxal 5'-phosphate (PLP), the PLP-binding residue Lys(38), and the amino acids in the immediate environment of the pyridoxal cofactor. The current model suggests that each active site of Streptomyces ALR maintains a larger space and takes a more rigid conformation than that of Bacillus stearothermophilus ALR determined previously. Furthermore, we show that Streptomyces ALR results in a slow conversion to a final form of a pyridoxal derivative arising from either isomer of cycloserine, which inhibits the catalytic activity noncompetitively. In fact, the slow conversion is confirmed by the fact that each enzyme-bound cycloserine derivative, which is bound to PLP, takes an asymmetric structure.