Computational design of cephradine synthase in a new scaffold identified from structural databases
Computational design of cephradine synthase in a new scaffold identified from structural databases
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从结构数据库中确定的新支架中头孢拉定合酶的计算设计
DOI：
10.1039/c7cc02270k
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发表时间：
2017-07-11
影响因子：
4.9
通讯作者：
Zhu, Yushan
中科院分区：
文献类型：
--
作者：
Huang, Xiaoqiang;Xue, Jing;Zhu, Yushan
计算酶设计在从结构数据库中识别潜在的支架和从无到有地创造新的酶催化剂方面表现出优异的性能。利用活性位点匹配算法ProdaMatch，我们从红球菌中鉴定出一种新的支架可卡因酯酶。这对β-内酰胺类抗生素头孢拉定的水解酶有一定的活性(k(CAT)/K-m=0.018 M-1 S(-1))。鉴定的可卡因酯酶支架与已知的β-内酰胺合成酶，如青霉素G酰基酶或α-氨基酸酯水解酶具有较低的同源性(0.30%)，并能催化D-二氢苯甘氨酸甲酯与7-氨基去乙酰氧基头孢烷酸的缩合反应，通过动力学控制合成头孢拉定。借助于计算酶设计方案，预测了可卡因酯酶支架中数百个序列，以促进头孢拉定的催化活性。此外，实验还证实了一个突变体(F261T)使催化效率提高了10倍(kcat/kM=0.193M(-1)S(-1))，这表明这种新型支架可卡因酯酶可能被重新设计，成为一种工业上有用的头孢拉定合成酶。
Computational enzyme design exhibits excellent performance for identifying potential scaffolds from structural databases and creating new enzymatic catalysts from naught. Using the active site-matching algorithm ProdaMatch, we identified a new scaffold cocaine esterase from Rhodococcus sp. that showed modest activity (k(cat)/K-m = 0.018 M-1 s(-1)) towards the hydrolysis of beta-lactam antibiotic cephradine. The identified cocaine esterase scaffold afforded low sequence identity (o30%) with the known b-lactam synthases, such as penicillin G acylase or alpha-amino acid ester hydrolase, and was able to catalyze the condensation reaction between D-dihydrophenylglycine methyl ester and 7-aminodesacetoxycephalosporanic acid to produce cephradine via a kinetically controlled synthesis. By virtue of the computational enzyme design protocol, hundreds of sequences were predicted in the cocaine esterase scaffold to promote the catalytic activity towards the hydrolytic reaction of cephradine. Moreover, a single mutant (F261T) was experimentally confirmed to have improved the catalytic efficiency by ten times (kcat/Km = 0.193M(-1) s(-1)), indicating that the novel scaffold cocaine esterase may be potentially redesigned to become an industrially useful cephradine synthase.