紫红红球菌9α-羟化酶loop对酶活性的影响

摘要

3-甾酮-9α-羟化酶是转化雄甾-4-烯-3,17-二酮生成9α-羟基雄甾-4-烯-3,17-二酮的关键酶,其加氧酶组分在底物进入酶活性中心入口处,存在一个由16个氨基酸构成的柔性 loop.为了明晰 loop对酶活性的影响,利用定点突变技术对 loop上结构变化较大的氨基酸进行研究,解析出了各氨基酸的作用,T224、N225、Y226与周围α,5螺旋和 β折叠存在一定相互作用,共同调节底物通道与底物运送能力;D227和 D228起到支撑固定 loop结构的作用,使T224-Y226能与其周围的氨基酸产生相互作用.这为进一步采用蛋白质工程改造9α-羟化酶提供了理论依据.%3-ketosteroid-9α-hydroxylase is the key enzyme that transforms androst-4-ene-3,17-dione into 9α-hydroxyan-drost-4-ene-3,17-dione.There exists a flexible loop which consists of 16 amino acids in front of the entrance of the active center of its oxygenase component.To understand the influence of the loop on the enzyme activity,site-directed mutagenesis was used to investigate the amino acids of the loop which moved remarkably in the whole protein structure.As a result, T224,N225 and Y226 were learned to adjust the substrate channel and transport the substrate with their surroundingα5-helix and β-sheet amino acids,while D227 and D228 played cerlain roles in supporting and fixing the loop,allowing T224-Y226 to interact with their surrounding amino acids.This work provided theoretical foundations for further research on 9α-hydroxylase modification through protein engineering.