Study On The Complex Structures Of Metal-β-Lactamase VMB-1 And Various β-Lactam Antibiotics

In recent years,due to the abuse of various types of antibiotics,more and more drug-resistant bacteria appear in clinic.The fatality rate caused by drug-resistant bacteria is also increasing,which has attracted worldwide attention.Specially,β-lactamase,as a drug-resistant"weapon"of most drug-resistant bacteria,has arised widespread attention.VMB-1（vibrio metallo-β-lactamase 1）is a novelβ-lactamase discovered in food-borne Vibrio alginolyticus recently.According to the type of hydrolyzed antibiotic and the characteristics of protein structure,VMB-1 is classified into subclass B1 of metallo-β-lactamases（MBLs）.VMB-1 is encoded by gene bla _VMB-1,located in an integron-bearing,highly transmissible Inc C plasmid,named p VB1796.p VB1796 can spread drug resistance by efficiently transferring bla _VMB-1 to other Gram-negative bacteria through conjugation experiments.In the near future,it is possible that strains carrying this gene will become drug resistant.In this paper,we aimed to study the hydrolyzing mechanism of VMB-1 by using protein crystallography techniques.1)According to the sequence analysis of bla _VMB-1,a VMB-1 protein expression plasmid was constructed for His-tagged protein expression in the prokaryotic system,and soluble expression of recombinant VMB-1 was obtained after the optimization of the induction temperature and the IPTG concentration;2)We carried out crystallization screening using the obtained protein with higher than 95%purity,and found that regular single crystals were grown under two conditions.On the basis of these conditions,co-crystallization of VMB-1 with penicillin G,ampicillin and cephalexin were attempted,respectively.After optimization,crystals with good size and shape were obtained.And then the crystals of VMB-1 were soaked with cefuroxime and meropenem,respectively;3)The above complex crystals were brought to the Shanghai Synchrotron Radiation Facility（SSRF）and Guizhou University for X-ray data collection.All diffraction data were processed using HKL3000 or XDS with satisfactory resolutions.Due to the binding restriction of substrate in VMB-1 crystals,only the co-crystal structure of VMB-1-meropenem has been solved,from which we realized that:i)VMB-1is very similar to most of metallo-β-lactamases in their overall structures,all adopting anαβ/βαfolding pattern,an active site containing two zinc ions;ii)Zn1 is ligated to three histidine residues,His99,His101,and His161,while Zn2 is coordinated with Asp103,Cys180,and His222,all these metal-ligands play irreplaceable roles for the catalytic activity of VMB-1;iii)Asn187 and Lys183 form hydrogen bonds with meropenem and thus stabilize the EP complex.Based on the catalytic characteristics of metallo-β-lactamases,the crystal structure of VMB-1-meropenem captured in this study was the EP complex.As for the co-crystal structures with over antibiotics,we found that the reason for the restriction of substrate binding in the VMB-1 crystal was that packing of the two molecules in the asymmetric unit led to narrowing of the pocket containing the active site,and thus hindered substrate binding.In this thesis,we determined the crystal structure of VMB-1 in complex with hydrolyzed meropenem,which laid a foundation for further studies of the hydrolyzing mechanism of VMB-1 and might provide an important basis for the research and development of MBL inhibitors.