Structural And Functional Studies Of Two Integral Membrane Enzymes Involved In Phospholipid And Sterol Metabolism

Glycerol 3-phosphate acyltransferase(PlsY)is a transmembrane protein located on bacterial cell membrane,which spans membrane for several times.PlsY is widespread in Gram-positive bacteria,but lacks eukaryotic homologs.PlsY catalyses the essential step in bacterial phospholipid biosynthesis by acylating the 1-OH of glycerol 3-phosphate using acyl-phosphate as the acyl donor to form lysophosphatidic acid.We obtained PlsY protein crystals in lipid cubic phase,and solved its high resolution(1.48?)crystal structure by X-ray crystallography.The structure showed that its seven-transmembrane fold with a relatively rigid V-shaped active site.We also obtained structures of PlsY with substrates or products bound.Substrate-and product-bound structures uncover the atomic details of the active site.Based on the structures,we proposed a‘substrate-assisted catalysis'mechanism.Unlike other acyltransferases,PlsY does not require a proteinaceous catalytic base to complete the catalysis.We also established in vitro activity assay,and several constants(including the relationship between protein concentration and enzyme activity,K_m of the two substrates,IC50 of the product to inhibit PlsY,the effects of temperature and p H on enzyme activity,etc.)of PlsY were determined.Structure-based mutagenesis and kinetic assays provided support for the catalytic mechanism.Using in vitro high-throughput activity assay in detergent micelles,we evaluated 88 compounds from structure-based docking and obtained a compound with the IC50 of 13?M.The result paves the way to antimicrobial discovery to fight multi-drug resistance.3-Hydroxy-3-Methylglutaryl-CoA reductase(HMGCR)is a rate limiting enzyme in cholesterol biosynthesis,and it is the central regulatory point of cholesterol biosynthesis pathway.HMGCR is located on the endoplasmic reticulum membrane,and both its transmembrane domain and catalytic domain are highly conserved.HMGCR is one of the most highly regulated enzymes in nature,and multiple levels(gene transcription,m RNA translation,protein degradation,enzyme activity)of the enzyme are regulated by negative feedback,which ensures a normal cellular cholesterol level.In this project,the transmembrane domain of human HMGCR was expressed and purified,but there was serious aggregation.Crystals were extensively screened,however,no protein crystals were obtained.In order to improve the stability,the apparent T_m of the transmembrane domain of HMGCR was increased from 41?to 69?by screening thermal stable mutants.The in vivo experiment showed that,like the wild type,the thermostable mutant(YN500)we obtained could be degraded by ubiquitination degradation.I also screened the synthetic single domain antibody(sybody)of YN500to stabilize protein conformation,and to aid crystallization.The screening of thermostable mutants and sybody improved the compositional stability and conformational stability of HMGCR transmembrane domain respectively,which lay a preliminary foundation for future structural biology studies.