Study On Sequence-structure-function Relationship Of Metallo ?-lactamases For Antibiotics Resistance Using Bioinformatic Methods

Metallo-?-lactamases(MBLs)compose the molecular group 3 according to the Bush-Jacoby-Medeiros functional classification and class B of Ambler.In recent years,many new enzymes of this class have been described and the sequences of the corresponding genes have been determined.In fact,most metallo-?-lactamases are broad-spectrum enzymes which also hydrolyze a variety of penicillins and cephalosporins even carbapenems.Although commercialized inhibitors of active site serine ?-lactamases are currently used in coadministration with antibiotic therapy,no clinically useful inhibitors of metallo?-lactamases have yet been discovered.The production of metallo ?-lactamases bacteria become recognized as multi-drug resistant pathogens.And it is becoming a major problem in clinical treatment increasingly.In this thesis,we used bioinformatic methods studying on sequence-structure-function relationship.of metallo(3-lactamases.A large number of MBLs sequences have been doing sequence homology analysis.Then we built phylogenetic trees to identify the evolutionary relationship between MBLs.Created models of unknown protein structures of MBLs by homology modeling.Finally,base on the sequence alignment and the structural superposition of protein structures,combined with published kinetic parameters of MBLs,analyse the protein structures and the amino acids mutants.In many MBLs types,we selected the typical Class B1 MBLs,the VIM-type,IMP-type and the IND-type.These amino acid sequences were carried out sequences alignment and homology analysis to construct phylogenetic trees.MBLs sequence alignment results showed that most of the tested MBLs have a generally conservative HFHDD motif,but NDM-1 has a very unique HAHQD motif.It possibly is the reason NDM-1 has different affinity to ?-lactam antibiotics compare with VIM-type and IMP-type.Protein structure analysis showed that:First,some MBLs amino acid residues display the highly conservative and act as an important role in the functional residues in protein such as Zn ligands(Znl ligands:His116?His118?His196;Zn2 ligands:Asp120?Cys221?His263)and Asn233(stable hydrolysis intermediates);Second,because Loop1 play an important role of connection with the substrate,the change of the Loop1 may impact on the interaction between the protein and substrate;Third,amino acid residues replacements in Loop3 affected the substrate hydrolysis,such as the VIM-2 sequence of the Loop3 His224Tyr,Ser228Arg two replacements lead improvement of carbapenems hydrolysis efficiency;Fourth,amino acid residues replacements near the active center,such as IMP-type Ser262Gly and IND-type Glu265Asp,which affect the hydrolysis of the substrate specificity.After all above analysises,we obtained a series of catalytic essential residues provide the basis for the detection of drug-resistant bacteria,and also provide the basis for the design of enzyme inhibitors.