中国动物传染病学报2024，Vol.32Issue(2)：1-11,11.
猪肺炎支原体LAP的结构预测及其抑制剂Bestatin对支原体生长的影响
Structural Modeling of Mycoplasma hyopneumoniae LAP and Inhibition Effect of Bestatin on Mycoplasma Growth
摘要
Abstract
Leucine aminopeptidase(LAP)is a ubiquitously founded metallopeptidase,playing important roles in amino acid metabolism.LAPs have irreplaceable roles in cell maintenance,growth and development.Previous research has showed that when the enzyme activity of LAP is inhibited,the growth of microorganism is stopped.In this study,we took Mycoplasma hyopneumoniae as a model to figure out its physical and chemical properties and to study the influence of LAP inhibitor bestatin on the growth of Mycoplasmas.Firstly,we carried out a sequence homology analysis by using Clustal Omega to confirm the conservation of LAP substrate binding sites.GST tag was used to increase the solubility of LAP proteins.GST affinity chromatography and gel filtration were used to purify LAP.Circular dichroism was carried out to determine the secondary structure of LAP.Homology modeling was used to build LAP structural model.Bestatin inhibition assay was carried out to test its influence to the growth of mycoplasmas.The results showed that Mycoplasma LAPs had almost all of the key residues for enzyme function in spite of low sequence identity.Mycoplasma hyopneumoniae LAP was expressed and purified as GST tag fusion proteins.When the GST tag was removed,LAP proteins were eluted at about 12ml on gel filtration,corresponding to molecular weight of a hexamer.Circular dichroism of LAP showed that it had a correct secondary structure.Modeling of LAP by Alpha fold 2 exhibited that LAP had the conserved structures and residues for leucine aminopeptidase enzyme activity.We also checked the inhibiting effect of bestatin to Mycoplasmas at different concentrations.Bestatin inhibition to Mycoplasmas was dose-dependent.Different Mycoplasmas had varying sensitivity to bestatin.The growth of Mycoplasmas was suppressed at the lowest bestatin concentration of about 6 μg/mL.The results from the present study would give insights for the development of new anti-mycoplasma drugs.关键词
猪肺炎支原体/亮氨酸氨肽酶/结构/活性位点Key words
Mycoplasma hyopneumoniae/leucine aminopeptidase/structure/active site分类
农业科技引用本文复制引用
陈蓉,冯志新,郝飞,谢星,赵琳,韦艳娜,张磊,王丽,熊琪琰,邵国青..猪肺炎支原体LAP的结构预测及其抑制剂Bestatin对支原体生长的影响[J].中国动物传染病学报,2024,32(2):1-11,11.基金项目
国家自然科学基金(31800160,31800161) （31800160,31800161）
省部共建国家重点实验室培育基地—江苏省食品质量安全重点实验室自主研究课题(2019sy004) （2019sy004）
