Study of Regulatory Mechanism of Catalysis by Cobalamin-dependent Enzymes
Grant-in-Aid for Scientific Research (C)
|Allocation Type||Single-year Grants|
|Research Institution||OKAYAMA UNIVERSITY|
TOBIMATSU Takamasa Okayama University, Engineering, Associate Professor, 工学部, 助教授 (30188768)
|Project Period (FY)
2002 – 2004
Completed(Fiscal Year 2004)
|Budget Amount *help
¥3,600,000 (Direct Cost : ¥3,600,000)
Fiscal Year 2004 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 2003 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 2002 : ¥2,200,000 (Direct Cost : ¥2,200,000)
|Keywords||Vitamin B12 / coenzyme B12 / radical enzyme / enzyme function / site-directed mutagenesis / active site / catalytic residue / X-ray crystallography / ジオールデヒドラターゼ / 基質結合部位 / グリセロールデヒドラターゼ|
1.X-ray analysis of glycerol dehydratase.
Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity and characterized. The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The structural features elucidated are quite similar to those of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.
2.Mutational analysis of active-site residues of diol dehydratase.
X-ray structure of diol dehydratase showed the seven amino acid residues interacting directly with active site K^+ and/or hydroxy group of substrate. To investigate the roles of these residues, alanine mutants of these residues were constructed and analyzed. The results
indicated the importance of H143 and E170 residues in the catalytic process. Role of the residue, H143, was examined further by constructing H143E,H143K,H143Q, and H 143L mutants. The results indicated the importance of hydrogen bond between His and 2-hydroxy group of propanediol for the catalytic reaction.
Role of two cobalamin-binding residues was investigated with site-directed mutagenesis. The results indicated that Sα224 is important for the activation of the Co-C bond of AdoCbl and for the prevention from inactivation during catalysis. The importance of Kβ135 for the binding of AdoCbl was also suggested.
3.Construction of H773G mutant of rat methionine synthase.
To construct mutant methionine synthase to which methylcobalamin was bound in base-on mode, His773 to Gly mutation was introduced into methionine synthase gene by site-directed mutagenesis. The method of high-level expression of rat methionine synthase in insect cells using a baculovirus expression system was tried to get the mutant methionine synthase, however, expression of mutant protein failed. Less
Research Products (6results)