Culbertson Justin E, Chung Dong hee, Ziebart Kristin T, Espiritu Eduardo, Toney Michael D
†Department of Chemistry, University of California, Davis, Davis, California 95616, United States.
‡Department of Chemistry, Oregon State University, Corvallis, Oregon 97331-4003, United States.
Biochemistry. 2015 Apr 14;54(14):2372-84. doi: 10.1021/acs.biochem.5b00013. Epub 2015 Apr 2.
The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS.
分支酸酶在细菌、真菌、寄生虫和植物中具有核心重要性，而在哺乳动物中不存在，这使得它们成为抗菌剂和除草剂的有吸引力的靶点。其中两种酶，邻氨基苯甲酸合酶（AS）和氨基脱氧分支酸合酶（ADCS），在结构和机制上相似。第一个催化步骤，即C2位的胺化，在它们之间是共同的，但AS还催化丙酮酸消除，将胺化中间体芳构化为邻氨基苯甲酸。尽管之前有过尝试，但尚未报道过将缺乏丙酮酸消除能力的酶转化为具有消除能力的酶。Janus是一种用于预测同源酶功能相互转化所需突变的生物信息学方法，被用于预测将ADCS转化为AS的突变。对Janus预测的突变文库进行了遗传筛选。在基本培养基上生长的AS缺陷型大肠杆菌菌株的互补导致了几个ADCS突变体，与野生型AS在2天内生长相比，这些突变体在6天内允许生长。纯化的突变酶催化分支酸向邻氨基苯甲酸的转化速率约为野生型ADCS催化分支酸向氨基脱氧分支酸转化速率的50%。突变的残基不与底物接触。分子动力学研究表明，丙酮酸消除受C2位胺化中间体构象的控制。催化消除的酶有利于赤道构象，该构象将C2-H呈现给保守的活性位点赖氨酸（Lys424）进行去质子化，并使立体电子活化最大化。通过显示溶剂衍生的质子掺入丙酮酸甲基以及通过溶剂动力学同位素效应研究AS催化的丙酮酸消除，证实了AS和水杨酸合酶中丙酮酸消除的酸碱催化作用。
