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Structural and Functional Studies of Saccharomyces Cerevisiae Aldo-keto Reductase Gre2

Author: BaoZhangZhi
Tutor: ChenYuXing
School: University of Science and Technology of China
Course: Biochemistry and Molecular Biology
Keywords: Saccharomyces cerevisiae aldo-keto reductase crystal structure substrate combination pocket -SDR superfamily complex structure
CLC: Q936
Type: Master's thesis
Year: 2011
Downloads: 26
Quote: 0
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Abstract


Most in vivo aldehydes and ketones are produced when the organism is stimulated by some kinds of stresses from the external environment. They can interefere the normal metabolic activities, or poison the organism by themselves. The organism has some mechanisms to reduce the influences of these stress-stimulated activities, by reducing the accumulation of the nonessential or harmful materials. Some proteins that called stress-responsive proteins are produced to conteract the extremely conditions.In this paper, the protein Gre2 we studied belongs to this kind of stress-responsive proteins. It is a kind of Saccharomyces cerevisiae oxidoreductase and possesses the reductive activity towards aldehydes and ketones. These oxidoreductases are grouped into four protein superfamilies: long-chain alcohol dehydrogenase superfamily, shortchain dehydrogenases/reductases(SDR) superfamily, medium-chain dehydrogenase superfamily(MDR) and aldo–keto reductase(AKR) superfamily. Gre2 belongs to SDR superfamily.As a kind of aldo-keto reductases towards aldehydes and ketones, Gre2 can catalyze the reduction reactions of many kinds of diketones,β-keto esters,α-keto esters, aliphatic and aromatic aldehydes, aliphatic ketones and hydroxy ketones. The microenvironment in S.cerevisiae can be changed by these reduction reactions. Gre2 can suppress the formation of filament and detoxify the methylglyoxal toxicity, via reduction reactions.Moreover, Gre2 can produce enantiopure hydroxy compounds in vitro, which can be served as valuable building blocks for the production of a variety of fine chemicals and pharmaceuticals.We constructed the Gre2-p28 recombination plasmid, and purified Gre2 recombinant-proteins. After crystal screening and optimization we obtained two kinds of Gre2 crystals for X-ray diffraction data collection: one for the apo form and another for the Gre2-NADPH complex.We resolved crystal structures of Gre2 in apo form at 2.7 ? and complexed with NADPH at 1.59 ? resolution by molecular replacement. Gre2 forms a homodimer, with each subunit containing a catalytic and a cofactor-binding domain.Comparision of the structure of Gre2 apo form and Gre2-NADPH complex, showed that upon NADPH binding to the interdomain cleft, the two domains shift towards each other to better fit for NADPH binding, and moreover to enable the binding of the substrate.As a member of SDR superfamily, the catalyze principle of Gre2 are similar to other SDR’s. Gre2 contains three conserved catalytic residues, Ser126, Tyr165 and Lys169. Ser126 and Tyr165 can form hydrogen bonds to the substrates, and donate proton in the reaction. Lys169 facilitates the reductive reaction via supplying the basic environment. We compared the pocket entrance of Gre2 and several SDRs of known three-dimensional structure. The entrance of carbonyl reductase from Sporobolomyces salmonicolor (PDB Code: 1Y1P), grape dihydroflavonol 4-reductase (PDB Code: 2C29), dihydroflavonol 4-reductase from Vitis vinifera (PDB Code: 2IOD) is very small, while that of Gre2 is relatively wide, which can accommodate diverse substrates. Apparently, above results provided a structural interpretation of the substrate specificity of the SDRs that the size of pocket openings decided the substrate specificity.

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