巴西苏木素对MRSA丙氨酸消旋酶抑制作用的初步研究
Preliminary studies on the inhibitory effect of Brazilinon MRSA alanine racemization enzyme
黎瑞 1陈泽慧 1周小仙 2余孟飞 3杨智芳 3李明哲3
作者信息
- 1. 遵义医科大学附属医院医学检验科,贵州遵义 563099;遵义医科大学检验医学院,贵州遵义 563099
- 2. 遵义医科大学附属医院医学检验科,贵州遵义 563099
- 3. 遵义医科大学检验医学院,贵州遵义 563099
- 折叠
摘要
目的 探讨巴西苏木素(BN)对耐甲氧西林金黄色葡萄球菌(MRSA)丙氨酸消旋酶(Air)的抑制作用.方法 PCR扩增MRSA丙氨酸消旋酶基因Alr,通过使用酶切和连接技术构建重组表达质粒pET-28a-Alr,然后将其转化至E.coil BL21(DE3)中,利用IPTG诱导表达Alr,并通过镍离子亲和层析法进行纯化;试验分为对照组MOCK(不作任何处理)、不同浓度BN组(8、16、32、64 μg/mL),分别测定不同浓度BN对Alr活性的影响;通过细胞热位移分析(CETSA)实验测定BN对Air蛋白热稳定性的影响;利用软件AutoDock Vina将BN与Air进行分子对接,预测两者之间的结合模式.结果 成功诱导表达纯化Alr蛋白,且其具备将L-丙氨酸外消旋为D-丙氨酸的活性.与对照组相比,不同浓度的BN均能够抑制Alr的活性,其中在64 μg/mL BN作用下,抑制率达到50％(P＜0.0001).CETSA实验结果显示,在55.9～59.5 ℃下BN处理后的Air蛋白热稳定性明显高于Mock组(P＜0.0001).分子对接结果显示BN与Air之间的结合能为-8.0 kcal/mol,与Air活性位点内的Gly221、Phe169、Arg219、Ser204、Ile222和Tyr43形成氢键,与其活性位点通道的Ile352、Tyr354形成π-π键及疏水作用,与Asn203形成疏水作用.结论 BN对Alr的活性具有抑制作用,可能是因为BN与Air蛋白活性位点处的结合能力相关.
Abstract
Objective This study aimed to investigate the inhibitory effects of Brazilin(BN)on the Alanine Racemase(Alr)of Methicillin-Resistant Staphylococcus aureus(MRSA).Methods The MRSA Alr gene(Alr)underwent PCR amplification,thena recombinant expression plasmid pET-28a-Alr was constructed using enzyme digestion and ligation techniques.The plasmid was transformed into E.coli BL21(DE3),and Alr expression was induced via IPTG.It was purified using nickel affinity chromatography.The experiment consisted of a MOCK control group with no treatment and several inhibition groups at different BN concentrations(8,16,32,64μg/ml)to evaluate the effects of BN on Alr activity.The effect of BN on Alr protein's thermal stability was ex-amined using Cell Thermal Shift Assay(CETSA).Molecular docking analyses utilizing Auto Dock Vina were ex-ecuted to predict the binding mechanism between BN and Alr.Results Alr was successfully induced and puri-fied,and the protein exhibited the activity of racemizing L-alanine to D-alanine.BN inhibited Alr activity at vari-ous concentrations compared to the control group,with a 50％inhibition observed at 64 μg/ml BN(P＜0.000 1).CETSA experiments confirmed that BN improved the thermal stability of Alr protein.Molecular docking demon-strated a binding energy of-8.0 kcal/mol between BN and Alr.Hydrogen bonds were formed between BN and Alractive sites Gly221,Phe169,Arg219,Ser204,Ile222,and Tyr43,π-π and hydrophobic interactions were formed between BN and AlrsiteIle352 and Tyr354(in the active site channel),and a hydrophobic interaction at Asn203 was also formed.Conclusion Brazilin(BN)displays inhibitory effects on Alr activity,potentially attrib-uting to its binding affinity with the Alr protein's active sites.
关键词
巴西苏木素/耐甲氧西林金黄色葡萄球菌/丙氨酸消旋酶/分子对接Key words
brazilian/methicillin-resistant staphylococcus aureus/alanine racemase/molecular docking引用本文复制引用
基金项目
贵州省科技厅科技项目(黔科合支撑2021一般034)
遵义市科技计划(遵市科合HZ字2021299)
遵义市科技计划(遵市科合 HZ 字202182)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据