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

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(Alr)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、64μg/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Alr蛋白热稳定性的影响;利用软件AutoDock Vina将BN与Alr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64μg/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Alr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Alr之间的结合能为-8.0 kcal/mol,与Alr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Alr蛋白活性位点处的结合能力相关。

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 examined using Cell Thermal Shift Assay(CETSA).Molecular docking analyses utilizing Auto Dock Vina were executed to predict the binding mechanism between BN and Alr.Results Alr was successfully induced and purified,and the protein exhibited the activity of racemizing L-alanine to D-alanine.BN inhibited Alr activity at various concentrations compared to the control group,with a 50%inhibition observed at 64μg/ml BN(P<0.0001).CETSA experiments confirmed that BN improved the thermal stability of Alr protein.Molecular docking demonstrated 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-πand 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 attributing to its binding affinity with the Alr protein's active sites.