Three Dimensional Structure Determination And Function Reaserch Of MpaA And AspR From Escherichia Coli O157

peptidoglycan(PG)is the essential component of the cell wall,which plays important roles in maintaining cell integrity,cell shape and osmotic pressure.The metabolism of PG includes anabolism and catabolism,and needs a series of enzymes.They work together,none is dispensable.In the pathogenic Escherichia coli O157,40%-50% of total PG will be reused per generation.The substrate of MpaA is the murein tripeptide L-Ala-?-D-Glu-meso-DAP,and MpaA is responsible for cleaving the amide bond between ?-D-Glu and meso-DAP.If MpaA activity is reduced or lost,it will affect the degradation of murein tripeptide,eventually inhibits the PG synthesis and affects the normal bacteria growth.In most bacteria,the D-amino acids are very scarce and are usually generated by the corresponding L-amino acids via the racemization of amino acid racemases.AspR belongs to amino acid racemase which can use L-Glu or L-Asp to generate the corresponding D-Glu or D-Asp by racemization.D-Glu is an indispensable ingredient in the composition of PG.If the D-Glu synthesis is blocked,its content will be reduced,then the synthesis of peptidoglycan will be blocked,which finally affects the integrity of the peptidoglycan and its normal growth,seriously causes cell death.In this paper,we have determined the three-dimensional structure and clarified the relationship between structure and function of MpaA and AspR in the pathogenic Escherichia coli O157.By cloning the genes and constructing the expression vectors,purifing and crystallizing the recombinant proteins,collecting the high-resolution crystal diffraction data,and determinating the three-dimensional structures of the two proteins to clarify the relationship between structure and function of MpaA and AspR respectively.MpaA and AspR work as potential targets for small molecule drugs,these results of this study will provide structural data for the development of novel small molecule drugs.The crystal structure of MpaA resolution is 2.6 ?.The final model of MpaA consists of two protein molecules in the asymmetric unit,each with a single Zn2+ ion.The MpaA monomer consists of 7 ?-helices and 10 ?-strands and possesses the common ?/? fold of metallo carboxypeptidases(MCP).Based on the analysis of amino acid sequence conservation: in the active centers the catalytic amino acids were highly conserved in the MpaA,Vh-MpaA and CPA1,and Zn2+ ion coordinated amino acid are completely conserved,which means that MpaA has the same catalytic reaction mechanism with the metallo carboxypeptidase superfamily.MpaA contains two important loop structures: Tyr133-Asp143 loop and Val204-Thr211 loop.The Tyr133-Asp143 loop is flexible and works as a lid cover the groove,which is responsible for regulating the substrate Mtp entry and binding;The Val204-Thr211 loop belongs to ordered region and is responsible for the substrate binding specificity.The crystal structure of apo-AspR resolution is 1.6 ?.There is one protein molecule in each asymmetric unit.The monomeric protein molecule contains two domains: the N terminal domain(N1-C101 and N216-C228)and the C terminal domain(N102-C215).Each domain is ?/? fold.Additionally we determined the crystal structures of AspR in complexes with its substrate L-Asp and product D-Asp at 1.76 ? and 1.59 ? respectively.No open–closed conformation changed when the substrate/ product binds to AspR.There is an unique catalytic pair Thr83-Cys197 in the active site.In the AspR-L-Asp complex,the ?-C from L-Asp is directed toward to the catalytic amino acid Cys197-thiol group,while the ?-C from D-Asp is directed toward to the catalytic amino acid Thr83-hydroxyl group in the AspR-D-Asp complex.Combining with the competitive activity of Cys-thiol group and Thr-hydroxyl group,these results well explain the single-track racemization mechanism of AspR.