Mechanism of Enzymatic Fluorination in Streptomyces cattleya
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- 作者：Xiaofeng Zhu ; David A. Robinson ; Andrew R. McEwan ; David O'Hagan ; James H. Naismith
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：November 28, 2007
- 年：2007
- 卷：129
- 期：47
- 页码：14597 - 14604
- 全文大小：463K
- 年卷期：v.129,no.47(November 28, 2007)
- ISSN：1520-5126
文摘
Recently a fluorination enzyme was identified and isolated from Streptomyces cattleya, as thefirst committed step on the metabolic pathway to the fluorinated metabolites, fluoroacetate and 4-fluorothreonine. This enzyme, 5'-fluoro-5'-deoxy adenosine synthetase (FDAS), has been shown to catalyzeC-F bond formation by nucleophilic attack of fluoride ion to S-adenosyl-L-methionine (SAM) with theconcomitant displacement of L-methionine to generate 5'-fluoro-5'-deoxy adenosine (5'-FDA). Althoughthe structures of FDAS bound to both SAM and products have been solved, the molecular mechanismremained to be elucidated. We now report site-directed mutagenesis studies, structural analyses, andisothermal calorimetry (ITC) experiments. The data establish the key residues required for catalysis andthe order of substrate binding. Fluoride ion is not readily distinguished from water by protein X-raycrystallography; however, using chloride ion (also a substrate) with a mutant of low activity has enabledthe halide ion to be located in nonproductive co-complexes with SAH and SAM. The kinetic data suggestthe positively charged sulfur of SAM is a key requirement in stabilizing the transition state. We propose amolecular mechanism for FDAS in which fluoride weakly associates with the enzyme exchanging two watermolecules for protein ligation. The binding of SAM expels remaining water associated with fluoride ion andtraps the ion in a pocket positioned to react with SAM, generating L-methionine and 5'-FDA. L-methioninethen dissociates from the enzyme followed by 5'-FDA.