Ogura Ken-ichi, Kunita Shin-ichi, Mori Koichi, Tobimatsu Takamasa, Toraya Tetsuo
Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Japan.
FEBS J. 2008 Dec;275(24):6204-16. doi: 10.1111/j.1742-4658.2008.06745.x. Epub 2008 Nov 7.
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 Seralpha224, and of 6-NH(2), N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the epsilon-NH(2) group of Lysbeta135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Seralpha224 and Lysbeta135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Salpha224A, Salpha224N and Kbeta135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kbeta135A, Kbeta135Q and Kbeta135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the beta135 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 Salpha224A and Salpha224N mutants showed a k(cat)/k(inact) value that was less than 2% that of the wild-type, whereas for Lysbeta135 mutants this value was in the range 25-75%, except for the Kbeta135E mutant (7%). Unlike the wild-type holoenzyme, the Salpha224N and Salpha224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Seralpha224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Salpha224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5'-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.
二醇脱水酶 - 腺苷基戊基钴胺素复合物的X射线结构表明，腺苷钴胺素的腺嘌呤部分通过N3与Serα224的侧链OH基团以及6 - NH₂、N1和N7与其他残基的主链酰胺基团之间的氢键作用，锚定在酶的腺嘌呤结合口袋中。在Lysβ135的ε - NH₂基团与钴胺素的磷酸基团之间形成了一个盐桥。为了评估腺嘌呤锚定和离子配对的重要性，制备了二醇脱水酶的Serα224和Lysβ135突变体，并研究了它们的催化特性。Serα224A、Serα224N和Kβ135E突变体的活性分别为野生型酶的19% - 2%，而Kβ135A、Kβ135Q和Kβ135R突变体保留了野生型活性的58% - 76%。β135残基处正电荷的存在增加了对钴胺素的亲和力，但对催化不是必需的，而在该处引入负电荷则阻止了酶与钴胺素的相互作用。Serα224A和Serα224N突变体的k(cat)/k(inact)值小于野生型的2%，而对于Lysβ¹35突变体，该值在25% - 75%范围内，Kβ135E突变体除外（7%）。与野生型全酶不同，Serα224N和Serα224A全酶在无底物时对氧气的敏感性非常低。这些发现表明Serα224对于钴 - 碳键的活化以及防止酶失活很重要。在催化过程中Serα224A全酶失活时，积累了钴（II）钴胺素，并且EPR中由于有机自由基产生的微量双峰信号消失。腺苷基团形成了5'-脱氧腺苷，并且脱辅酶本身未受损。因此，这种失活被认为是基于机制的失活。
