Wu Qingquan, Liu Yung-Nan, Chen Huawei, Molitor Erich J, Liu Hung-wen
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Biochemistry. 2007 Mar 27;46(12):3759-67. doi: 10.1021/bi602352g. Epub 2007 Feb 27.
CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (E1), which catalyzes C-3 deoxygenation of CDP-4-keto-6-deoxyglucose in the biosynthesis of 3,6-dideoxyhexoses, shares a modest sequence identity with other B6-dependent enzymes, albeit with two important distinctions. It is a rare example of a B6-dependent enzyme that harbors a [2Fe-2S] cluster, and a highly conserved lysine that serves as an anchor for PLP in most B6-dependent enzymes is replaced by histidine at position 220 in E1. Since alteration of His220 to a lysine residue may produce a putative progenitor of E1, the H220K mutant was constructed and tested for the ability to process the predicted substrate, CDP-4-amino-4,6-dideoxyglucose, using PLP as the coenzyme. Our data showed that H220K-E1 has no dehydrase activity, but can act as a PLP-dependent transaminase. However, the reaction is not catalytic since PLP cannot be regenerated during turnover. Reported herein are the results of this investigation and the implications for the role of His220 in the catalytic mechanism of E1.
CDP-6-脱氧-L-苏式-D-甘油-4-己酮糖-3-脱水酶（E1）在3,6-二脱氧己糖的生物合成中催化CDP-4-酮-6-脱氧葡萄糖的C-3脱氧反应，它与其他依赖维生素B6的酶有一定程度的序列同源性，不过存在两个重要区别。它是罕见的含有[2Fe-2S]簇的依赖维生素B6的酶的例子，并且在大多数依赖维生素B6的酶中作为磷酸吡哆醛（PLP）锚定位点的一个高度保守的赖氨酸在E1中被第220位的组氨酸取代。由于将His220改变为赖氨酸残基可能产生E1的假定祖先，因此构建了H220K突变体，并测试了其以PLP作为辅酶处理预测底物CDP-4-氨基-4,6-二脱氧葡萄糖的能力。我们的数据表明，H220K-E1没有脱水酶活性，但可以作为依赖PLP的转氨酶起作用。然而，该反应不具有催化性，因为在周转过程中PLP无法再生。本文报道了这项研究的结果以及His220在E1催化机制中的作用的相关推论。
