Summary of Research Project Results under JSPS FY2001
"Research for the Future Program"



1.Research Institution Kyoto University
 
2.Research Area Integrated Fields
 
3.Research Field Production of Novel Useful Substances through Integration of Life Sciences and Chemistry
 
4.Term of Project FY 1997 〜 FY 2001
 
5.Project Number 97I00302
 
6.Title of Project Fine Enzymatic Syntheses of Useful Compounds

7.Project Leader
Name Institution,Department Title of Position
Sakayu Shimizu Kyoto University, Graduate School of Agriculture Professor

8.Core Members

Names Institution,Department Title of Position
Hiromichi Ohta Keio University, Department of Chemistry Professor
Hidehiko Kumagai Kyoto University, Graduate School of Biostudies Professor
Tanetoshi Koyama Tohoku University, Institute of Multidisciplinary Research for Advanced Materials Professor

9.Summary of Research Results

 We have developed a novel bioreduction system, in which Escherichia coli transformant cell co-expresing an NAD(P)H-dependent carbonyl reductase gene and that of glucose dehydrogenase as a cofactor regenerator are used as a catalyst, for asymmetric reduction of prochiral carbonyl compounds to corresponding chiral alcohols. This bioreduction system is applicable to the production of many useful chiral alcohols, by replacing the carbonyl reductase gene for appropriate reductase genes. We have also succeeded to develop some new reactions, such as hydrolysis of nitriles, asymmetric decarboxylation of α-arylmethylmalonate, which are useful for the synthesis of chiral molecules. Based on the mechanism, we have succeeded in the inversion of enantioselectivity of the decarboxylase via site-directed mutagenesis. The derivatives of biologically active peptides having sugar chain were chemo-enzymatically synthesized by Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M). The synthesized glycopeptides were those of peptide T, Substance P, etc. They were resistant to the attack of proteases and retained their original activities. The cDNA of Endo-M was cloned and overxpressed in Candida boidonii. The high mannose type sugar chain of bovine serum RNaseB was remodeled to complex type sugar chain. These results strongly indicate the important role of the enzyme in the development of glycotechnology in future. The structural genes for many kinds of prenyltransferases that catalyze (E)-type prenyl chain elongation had been identified and characterized. However, no information has been available about the structures of (Z)-prenyl diphosphate synthases until our isolation of the gene for the Micrococcus luteus undecaprenyl diphosphate (UPP) synthase. The amino acid sequence of the (Z)-prenyl diphosphate synthase is totally different from those of (E)-prenyl chain elongating enzymes. Moreover, the crystal structure of the UPP synthase has been determined at 2.2 Å resolution as the first three dimensional structure among cis-prenyl chain elongating enzymes. This enzyme shows a novel protein fold, which is completely different from those for the enzymes relating to isoprenoid biosynthesis.

10.Key Words

(1)Enzymatic reduction system、(2)Biocatalyst、(3)Alteration of enzyme functions
(4)Aymmetric decarboxylation、(5)Conversion of synthetic substrates、(6)Endo-β-N-acetylglucosaminidase
(7)Chemo-enzymatic synthesis of glycopeptides、(8)Remodeling of sugar chain of glycoprotein、(9)Prenylchain elomgating enzyme


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