The Structural Biology Research Of IAH1 And VMA7 From Saccharomyces Cerevisiae

(I) The crystallographic and functional research on IAH1 from Saccharomyces CerevisiaeIAH1 from Saccharomyces cerevisiae is an esterase that catalyzes the hydrolytic cleavage of isoamyl acetate, which greatly determines the flavor and quality of wine during alcoholic fermentation. Here we report the crystal structure of IAH1 at 1.60? resolution. By examining the crystal structure, we have identified its enzymatic classification and further explored the structural basis of substrate specificity. The overall structure of IAH1 adopts a folding pattern similar to the canonical SGNH-superfamily topology. However, some features also distinguish it from other SGNH-hydrolases, such as a long additional C-terminus. This additional C-terminus, which is concerned with homodimer forming, involves in the restriction of access to the active site of its dimer mate monomer and is found to play an important role in the substrate specificity. When the C-terminus was deleted, the truncated variant represented much broader substrate specificity that shared characteristic of a lipase. Within the structure of IAH1, the active hole can be divided into three levels that each has their own respective function. Because of steric hindrance effect, we conjecture that the longer acyl chain of substrate is inclined to remain on the upper region of the active hole. Therefore, the residues Trp129, Lys133, and Trp226 (from the dimer mate monomer) might serve as key residues to provide the necessary hydrophobic environment for binding the aliphatic chain of the substrate.(II) Crystallization and preliminary crystallographic analysis of VMA7 from Saccharomyces CerevisiaeThe pH of intacellular compartments and the extracellular environment is a carefully controlled parameter that affects many cellular processes. In eukaryotic cells, at the heart of regulating pH are V-ATPases, which are composed of dozens of subunits. These subunits are organized into two domains, a peripheral V1 domain and a membrane-embedded V₀ domain. The two domains are connected by a central stalk, composed of subunits D and F of V1and subunit d of V₀. Here, We cloned the V-ATPase F subunit (VMA7) gene from Saccharomyces Cerevisiae genomic DNA and insert it into multiple cloning site 1 of pETDuet vector, expressed the VMA7 protein in E.coli BL21(DE3) strain. The recombinant protein was then purified by using a Nickel-chelating Resin and a Gel filtration column. We also used the hanging-drop vapor diffusion method to screen the initial crystallization conditions. The work of optimizing the crystallization conditions is still going on.