Characteristics and crystal structure of bacterial inosine-5′-monophosphate dehydrogenase
Characteristics and crystal structure of bacterial inosine-5′-monophosphate dehydrogenase
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DOI：
10.1021/bi982858v
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发表时间：
1999-04-13
期刊：
影响因子：
2.9
通讯作者：
Collart, FR
中科院分区：
文献类型：
--
作者：
Zhang, RG;Evans, G;Collart, FR
IMP脱氢酶（IMPDH）是催化GTP合成的第一步的必需酶。为评价IMPDH抑制剂作为抗微生物剂提供基础，我们表达并表征了来自致病性细菌化脓性链球菌的IMPDH。结果表明，S.化脓菌IMPDH与其它细菌IMPDH酶相似。然而，缺乏对霉酚酸的敏感性和NAD的K-m（1180 μ M）弥补了细菌和哺乳动物IMPDH酶之间的一些差异，使其成为抗微生物剂的有吸引力的靶标。为了评估这些差异的基础，我们在1.9埃下测定了细菌酶的晶体结构，底物结合在催化位点。使用硒代蛋氨酸取代的蛋白质和多波长异常（MAD）分析从阿贡先进光子源的结构生物学中心的波荡器光束线（19 ID）同步辐射获得的数据，确定结构。S.化脓性链球菌IMPDH是四聚体，其四个亚基通过晶体学4重轴相关。该蛋白由两个结构域组成：一个TIM桶结构域，体现了催化框架和一个胱硫醚β-合酶（CBS）二聚体结构域，迄今未知的功能。利用序列比对和晶体结构提供的信息，我们制备了几个位点特异性突变体，以研究各种活性位点区域在催化中的作用。这些变体暗示活性位点瓣作为一个必要的催化元素，并表明有显着差异的催化环境中的细菌和哺乳动物IMPDH酶。比较细菌IMPDH的结构与已知的真核生物的部分结构，将提供一个解释其独特的属性，并有助于特定的细菌IMPDH抑制剂的设计。
IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the K-m for NAD (1180 mu M) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 Angstrom with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione beta-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.