摘要
本研究旨在以猪肺炎支原体为代表,研究支原体LAP酶活中心的高级结构及其被抑制后对支原生长的影响。亮氨酸氨肽酶(LAP)是一类广泛存在于各类生物的氨基酸代谢酶,有研究表明病原微生物的LAP酶活中心被抑制后可以直接影响微生物的代谢和生长。本研究拟以猪肺炎支原体(Mhp)LAP为模型,研究其理化特性和高级结构,探索LAP抑制剂乌苯美司(Bestatin)对支原体生长的影响。本研究首先用Clustal Omega进行序列同源性比较,分析LAP活性位点在不同支原体中的保守情况。然后采用GST融合标签表达猪肺炎支原体LAP蛋白,通过GST亲和层析和分子筛层析纯化蛋白,圆二色谱测定LAP的二级结构,用Alpha fold 2预测三级结构。最后用Bestatin与猪、鸡、羊等不同种属的支原体共培养来观察其影响。结果表明虽然总体序列同源性不高,但是LAP酶活中心的关键位点在常见支原体相对保守。猪肺炎支原体LAP可以通过GST标签进行可溶性表达。酶切GST标签后,分子筛层析显示LAP是六聚体,圆二色谱表明LAP具有正确的二级结构。预测的LAP三级结构显示其具有保守的底物结合关键位点。Bestatin对不同种属支原体的生长均产生抑制,且呈现剂量依赖性,但在不同菌株间存在差异,最低在6μg/mL的浓度可通过抑制氨肽酶的活性抑制猪肺炎支原体的生长。该研究为新一代抗菌药物的研发提供了新的思路。
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 purifi ed 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 6μg/mL.The results from the present study would give insights for the development of new anti-mycoplasma drugs.
作者
陈蓉
郝飞
谢星
赵琳
韦艳娜
张磊
王丽
熊琪琰
邵国青
冯志新
CHEN Rong;HAO Fei;XIE Xing;ZHAO Lin;WEI Yanna;ZHANG Lei;WANG Li;XIONG Qiyan;SHAO Guoqing;FENG Zhixin(Key Laboratory for Veterinary Bio-Product Engineering,Ministry of Agriculture and Rural Affairs,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)
出处
《中国动物传染病学报》
CAS
北大核心
2024年第2期1-11,共11页
Chinese Journal of Animal Infectious Diseases
基金
国家自然科学基金(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)。
关键词
猪肺炎支原体
亮氨酸氨肽酶
结构
活性位点
Mycoplasma hyopneumoniae
leucine aminopeptidase
structure
active site
