Preliminary studies on the inhibitory effect of Brazilinon MRSA alanine racemization enzyme
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.
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