Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics

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• 作者：Vangmayee Sharma ; Yane-Shih Wang ; Wenshe R. Liu
• 刊名：ACS Chemical Biology
• 出版年：2016
• 出版时间：December 16, 2016
• 年：2016
• 卷：11
• 期：12
• 页码：3305-3309
• 全文大小：315K
• ISSN：1554-8937

文摘

Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine, can be genetically encoded using engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows a dramatic loss in the enzyme’s catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme.