2004 Fiscal Year Final Research Report Summary
Functional modification of amino-acid dehydrogenases by the combination of site-directed mutagenesis and evolutionary molecular engineering
| Project/Area Number |
15580079
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Shimane University |
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Principal Investigator |
SAWA Yoshihiro Shimane University, Faculty of Life and Environmental Science, Professor, 生物資源科学部, 教授 (70127489)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Amino acid dehydrogenase / Alanine dehydrogenase / Glutamate dehydrogenae / Aspartate dehydrogenase / L-aspartic acid |
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| Research Abstract |
One of the major goals of our studies has been to understand the structural basis of amino acid substrate specificity in amino acid dehydrogenase, and to apply such knowledge to the engineering of novel substrate or cofactor specificities. The three dimensional structure of aspartate aminotransferase from Phormidium lapideum has been determined for design of aspartate binding site by X-ray crystallography. The alanine dehydrogenase gene and glutamate dehydrogenase gene were chosen as templates for the mutagenesis. The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in E.coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined as a hexameric structure (M_r 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate requiring NADH and NAD^+ as cofactors, respectively. The enzyme showed low thermostability with T_m=41℃ due to low structural integrity of hexamer. To improve thermal stability of this enzyme, we performed error-prone PCR introducing random mutagenesis on cloned GluDH. Two single mutant enzymes Q144R and E27F were isolated from mutant library whose T_m values were 61℃ and 49℃, respectively. Furthermore, Q144R has a remarkably high k_<cat> value (435 s^<-1>) for amination reaction at 37℃ which is 1.3 times higher than that of wild type. Among the residues predicted to be important involving substrate recognition, Lys80,Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild type enzyme is specific for 2-oxoglutarate with k_<cat> value 344 s^<-1>, whereas G82K and M101S are specific for oxaloacetate with k_<cat> values 3.45 and 5.68 s^<-1>, which are 265 and 473 folds higher than those for 2-oxoglutarate, respectively. We are in progress to enhance the k_<cat> values of those mutant enzymes by using molecular evolutional engineering.
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