| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1996: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
I investigated the role of aldo-keto reductases which may involve in the metabolism of polycyclic aromatic hydrocarbons in animal tissues. In the course of this project, three dihydrodiol dehydrogenase [EC.1.1.2.? ] were found and purified in bovine liver cytosol. Among these enzymes, the results of substrate specificities, inhibitor sensitivities and immunological crossreactions with corresponding antibody suggested that two enzymes (DD1 and DD2) were identified with 3alpha-hydroxsteroid dehydrogenase and aldehyde reductase, respectively. However, these enzymological and immunological properties of the third enzyme (DD3) were unique among the dihydrodiol dehydrogenses which have been reported. The partial primary structure of DD3 has been reported and its important cysteine residue which may involves in the enzymatic regulation by redox balance. Based on this amino acid sequence, DNA probe was synthesized with PCR method. As a results of the plaque hybridization study, the DD3 cDNA wa
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s cloned from male adult bovine liver cDNA library. The DD3 cDNA carrying a size of 1339 bp (323 amino acids) inserted into the expression vector pET28a (pET28a-dd3). pET28a-dd3 was transformed into E.coli bacterial cells and the recombinant protein (rDD3) was expressed with IPTG.Overexpressed rDD3 was purified with one step method of nickel affinity column with high yield and quarity. According to the same method, the mutated rDD3s which were introduced their point mutations with megaprimer PCR method using mutated primers were produced in bacterial cells. The results of substrate specificities and inhibitor sensitivities of these 9 mutants (D50N,Y55F,Y55H,K84N,H117Q,H117Y,Y55F+H117Q,C145S and C193S) suggested the following roles of these amino acids in the DD3 activity. Computer simulation of 3D-structure suggests that DD3 has a typical alpha8, beta8-barrel structure and tAsp-50, Tyr-55, Lys-84, His-117 and Cys-193 may locate in the barrel and Cys-145 in the surface of structure.
1. Tyrosine-55 is an essential amino acid and may play an critical role in the transfer of hydride between coenzymes and substrates (proton relay) in the DD3. In the catalytic activity of DD3, the residue which may have an ability to involve in the hydride transfer should places at position 55 in the enzyme structure.
2. Both aspartate-50 and lysine-84 play an important role in the proton relay by stabilizing the intermediate (tyrosine-55, coenzyme and substrate) in the enzyme structure.
3. Histidine-117 being an important residue in the active site may involve in the recognition of substrates and may not involve directly in the proton relay.
4. The computer simulations and CD-spectrum studies of wild type and mutated DD3s suggested that the structural changes that affects the loss of activity.
5. Cystiene-193 which places in the cleft of coenzyme binding site of DD3 plays an important role in the regulation of DD3 activity, but, cysteine-145 did not have any role in the activity because it locates in the surface of the enzyme structure.
In conclusion, the study of this project disslove easily the important relationship between the enzymatic activity and the position of amino acid resdiues in the 3D-structure by the combination of site-directed mutagenesis and computer simulation of the structure of snzyme. Less
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