Single‐Turnover Kinetics Reveal a Distinct Mode of Thiamine Diphosphate‐Dependent Catalysis in Vitamin K Biosynthesis |
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Authors: | Mingming Qin Dr Haigang Song Xin Dai Chi‐Kong Chan Prof Wan Chan Prof?Dr Zhihong Guo |
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Affiliation: | 1. Department of Chemistry, The Hong Kong University of Science and Technology, Kowloon, Hong Kong;2. Present address: Division of Structural Biology, Wellcome Trust Centre of Human Genomics, University of Oxford, Oxford, UK;3. Environmental Science Program, The Hong Kong University of Science and Technology, Kowloon, Hong Kong |
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Abstract: | MenD, or (1R,2S,5S,6S)‐2‐succinyl‐5‐enolpyruvyl‐6‐hydroxycyclohex‐3‐ene‐1‐carboxylate (SEPHCHC) synthase, uses a thiamine diphosphate (ThDP)‐dependent tetrahedral Breslow intermediate rather than a canonical enamine for catalysis in the biosynthesis of vitamin K. By real‐time monitoring of the cofactor chemical state with circular dichroism spectroscopy, we found that a new post‐decarboxylation intermediate was formed from a multistep process that was rate limited by binding of the α‐ketoglutarate substrate before it quickly relaxed to the characterized tetrahedral Breslow intermediate. In addition, the chemical steps leading to the reactive post‐decarboxylation intermediates were not affected by the electrophilic substrate, isochorismate, whereas release of the product was found to limit the whole catalytic process. Moreover, these intermediates are likely kinetically stabilized owing to the low biological availability of isochorismate under physiological conditions, in contrast to the tight coupling of enamine formation with binding of the electrophilic acceptor in some other ThDP‐dependent enzymes. Together with the unusual tetrahedral structure of the intermediates, these findings strongly support a new ThDP‐dependent catalytic mode distinct from canonical enamine chemistry. |
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Keywords: | Breslow intermediate enzyme catalysis kinetics thiamine diphosphate vitamin K |
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