TAILIEUCHUNG - Báo cáo khoa học: Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis

The X-ray structure of the diol dehydratase–adeninylpentylcobalamin com-plex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Sera224, and of 6-NH2, N1 and N7 with main chain amide groups of other residues. | Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis Ken-ichi Ogura Shin-ichi Kunita Koichi Mori Takamasa Tobimatsu and Tetsuo Toraya Department of Bioscience and Biotechnology Graduate Schoolof NaturalScience and Technology Okayama University Japan Keywords adenine anchoring adenosylcobalamin coenzyme B12 diol dehydratase ion pairing Correspondence T. Toraya Department of Bioscience and Biotechnology Graduate Schoolof Natural Science and Technology Okayama University Tsushima-naka Okayama 700-8530 Japan Fax 81 86 251 8264 Tel 81 86 251 8194 E-mail toraya@ Received 4 September 2008 revised 8 October 2008 accepted 15 October 2008 doi The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Sera224 and of 6-NH2 N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the e-NH2 group of Lysb135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing Sera224 and Lysb135 mutants of diol dehydratase were prepared and their catalytic properties investigated. The Sa224A Sa224N and KP135E mutants were 19-2 as active as the wild-type enzyme whereas the KP135A KP135Q and KP135R mutants retained 58-76 of the wild-type activity. The presence of a positive charge at the P135 residue increased the affinity for cobalamins but was not essential for catalysis and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Sa224A and Sa224N mutants showed a kcat kinact value that was less than 2 that of the wild-type whereas for Lysb135 mutants this value was in the range 25-75 except for the KP135E mutant 7 . Unlike the wild-type holoenzyme the Sa224N and Sa224A holoenzymes showed very low

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