Characterization of the N-oxygenase AurF from Streptomyces thioletus
Characterization of the N-oxygenase AurF from Streptomyces thioletus
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DOI：
10.1016/j.bmc.2014.06.002
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发表时间：
2014-10-15
影响因子：
3.5
通讯作者：
Zhao, Huimin
中科院分区：
文献类型：
--
作者：
Chanco, Emmanuel;Choi, Yoo Seong;Zhao, Huimin
AurF催化对氨基苯甲酸的N-氧化为对硝基苯甲酸的生物合成抗生素金黄色。在这里，我们报告了AurF在优化条件下的表征，以探索其在生物催化中的潜在应用。以吩嗪甲硫酸盐(PMS)/NADH为酶介体体系，该酶的最适pH为5.5，比文献报道的酶活力高10倍。在优化的反应条件下，Km=14.7+/-1.1u M，k(CAT)=47.5+/-5.4min(-1)，a k(CAT)/K-m=3.2+/-0.4muM-1min(-1)。PMS/NADH和天然电子转移蛋白均能显著生成对羟基氨基苯甲酸中间体，而H_2O_2主要生成对硝基苯甲酸。丙氨酸扫描确定了重要活性部位残基的作用。根据AurF的晶体结构，对AurF的底物专一性进行了检验和合理化。动力学研究表明，K-m是AurF对替代底物活性的主要决定因素。(C)2014爱思唯尔有限公司。保留所有权利。
AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing similar to 10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a K-m of 14.7 +/- 1.1 mu M, a k(cat) of 47.5 +/- 5.4 min(-1) and a k(cat)/K-m of 3.2 +/- 0.4 mu M-1 min(-1.)PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H2O2 produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the K-m is the main determinant of AurF activity toward alternative substrates. (C) 2014 Elsevier Ltd. All rights reserved.