| Budget Amount *help |
¥8,700,000 (Direct Cost: ¥8,700,000)
Fiscal Year 2000: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 1999: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Research Abstract |
The flavin reductase of luminous bacterium has a close relation to a luminescence phenomenon, and the nitro reductase is related to mutation. In this research, we investigated both wild type and mutant of the flavin reductase (FRasel from luminous baderium Vivno fischerii) and the nitro reductase (NfsA from E Coli.) by X-ray crystallography. The purpose is to clarify mechanism of the enzyme reaction and substrate specificity on an atomic level.
We have already determined the crystal structure of wild type FRasel in 1.7Å resolution. In this research, the structures of the complex with inhibitors (dicoumarol, hydroxycoumarin, warfarin, etc.) were determined by X-ray crystallography. We also produced various mutants with replaced Phe124 because Phe124 participates in the activity of FRasel. And we performed enzyme reaction kinetics analysis using those mutants. Regarding the Phe124Ala mutant and the Phe124Trp mutant, X-ray crystallography of the mutant only and the complex with the inhibitor was performed. As a result, we could consider the residue expressing substrate specificity.
Although This enzyme family containing FRasel, NfsA, etc. has all of flavin reduction activity, nitro reduction activity and quinone reduction activity, we have made it clear that they are classified according to the substrate with the highest activity.
Regarding the NfsA, random mutation was introduced by PCR. These mutants were applied to activity screening, and some mutants showing activity were acquired. Among these, the mutants with Phe42 replaced by another 11 amino acids were created and the NADPH oxidization activity was analyzed. Consequently, it was shown that the benzene ring structure in a substrate joint pocket contributed to the specificity of NADPH.
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