Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica
Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica
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DOI：
10.1016/j.bbagen.2013.09.038
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发表时间：
2014-01-01
影响因子：
3
通讯作者：
Escalante-Semerena, Jorge C.
中科院分区：
文献类型：
--
作者：
Chan, Chi Ho;Newmister, Sean A.;Escalante-Semerena, Jorge C.
背景：钴酰胺的多样性源于核苷酸碱基的性质。烟酸单核苷酸酯（NaMN）：碱基磷酸核糖基转移酶（CobT）从不同的核苷酸碱基底物（例如，方法：对CobT酶家族的两个成员与各种底物碱基复合的结构研究以及酶变体的体内和体外活性分析进行了研究，以阐明对底物识别重要的关键氨基酸残基的作用。结果：肠道沙门氏菌CobT（SeCobT）酶的活性位点变体的体外和体内研究结果表明，催化可能不需要催化碱。这一想法得到了晶体结构分析的支持，晶体结构分析表明，两个谷氨酸残基的功能主要是保持酶的活性构象。鉴于这些发现，我们建议，在活性位点的基板的适当定位触发的攻击在C1核糖NaMN.Conclusion：是否需要一个催化基地的功能进行了讨论的框架内的酶活性的体外分析。此外，结构指导的定点诱变SeCobT拓宽了其底物特异性，包括酚类碱基，揭示可能的进化变化需要增加cobamide多样性，并进一步支持提出的机制为phosphoribosylation的酚substrate.General Significance：这项研究的结果揭示了关键的残基在CobT酶，有助于在自然界中的cobamides的多样性。(C)2013爱思唯尔有限公司版权所有。
Background: Cobamide diversity arises from the nature of the nucleotide base. Nicotinate mononucleotide (NaMN):base phosphoribosyltransferases (CobT) synthesize a-linked riboside monophosphates from diverse nucleotide base substrates (e.g., benzimidazoles, purines, phenolics) that are incorporated into cobamides.Methods: Structural investigations of two members of the CobT family of enzymes in complex with various substrate bases as well as in vivo and vitro activity analyses of enzyme variants were performed to elucidate the roles of key amino acid residues important for substrate recognition.Results: Results of in vitro and in vivo studies of active-site variants of the Salmonella enterica CobT (SeCobT) enzyme suggest that a catalytic base may not be required for catalysis. This idea is supported by the analyses of crystal structures that show that two glutamate residues function primarily to maintain an active conformation of the enzyme. In light of these findings, we propose that proper positioning of the substrates in the active site triggers the attack at the C1 ribose of NaMN.Conclusion: Whether or not a catalytic base is needed for function is discussed within the framework of the in vitro analysis of the enzyme activity. Additionally, structure-guided site-directed mutagenesis of SeCobT broadened its substrate specificity to include phenolic bases, revealing likely evolutionary changes needed to increase cobamide diversity, and further supporting the proposed mechanism for the phosphoribosylation of phenolic substrates.General Significance: Results of this study uncover key residues in the CobT enzyme that contribute to the diversity of cobamides in nature. (C) 2013 Elsevier B.V. All rights reserved.