Structural And Functional Studies Of Budding Yeast Mek1 In Complex With Hop1 And Deacetylase CmCBDA From Cyclobacterium Marinum

In the first part of the dissertation,we introduced the structure and function of the interaction between Hopl and Mekl,the lateral elements of synaptonemal complex,during Saccharomyces cerevisiae meiosis.Meiosis is a special cell division and an indispensable part of sexual reproduction.The normal progression of meiosis is critical for maintaining the stability of chromosome number and producing diverse offspring.Homologous recombination in the meiotic prophase I is an important process for achieving exchanges between parental genes.The successful progress of homologous recombination requires the participation of synaptonemal complex to achieve precise regulation of biological behaviors in the progress.The structural component Hopl of synaptonemal complex induces the localization and activation of the meiosis-specific kinase Mekl by the interaction between phosphorylated threonine at position 318 on Hopl SCD domain and the Mekl FHA domain.The activated Mekl phosphorylates a range of substrates to promote the repair templates bias to the non-sister chromatids of homologous chromosome,thereby enabling homologous recombination of meiosis and subsequent biological progress.How Mekl-FHA recognizes Hopl pT318 is vital for a deep understanding of Mekl function.The crystal structure of the apo Mekl-FHA and in complex with Hopl pT318 peptide were solved and presented herein.By analyzing the FHA domains in Protein Data Bank(PDB)and Mekl-FHA,we proposed a class of PSXS Motif located in the binding pocket.This kind of Motif shows some conservation on the three-dimensional structure and the substrate recognition mode.It was shown in the complex structure that Phe320 and Val321,the +2 and +3 residues of Hopl pT318,are important for the selective recognition of Mekl-FHA.Based on this rule,we identified the potential interacting sites of Mekl-FHA.The results of isothermal titration calorimetry(ITC)showed that the substracts without hydrophobic residues in pT+2 and pT+3 were not recognized by Mekl-FHA.Moreover,we identified in the fission yeast that Mekl-FHA recognizes Hopl pT270.Combined with the previously published literature,Mekl could recognize its own N-terminal pT15.We believe that fission yeast Mekl execute auto-activation in two ways,which is significantly different from the activation mode of budding yeast Mekl.Our experimental results better understand the differences in structure and function of the interaction between Mekl and Hopl during meiosis in S.cerevisiae and Schizosaccharomyces pombe.In the second part of the dissertation,we introduced the structural study of the Cyclobacterium marinum deacetylase CmCBDA.Osteoarthritis is a very common senile disease,affecting hundreds of millions of people worldwide.The main cause of senile osteoarthritis is the degradation of cartilage.One of the important ways to prevent and treat cartilage degradation is to take glucosamine products.Therefore,glucosamine products have become herbal and dietary supplements and prescription drugs that are favored by the global market.However,the defects in the industrial production process of glucosamine have caused an increase in the production cost of glucosamine products,resulting in high market prices and the inability of many patients to purchase.Up to date,the chanllenging step in the industrial production of glucosamine is the process of deacetylating the precursor acetylglucosamine(GlcNAc)to glucosamine(G1cN).CmCBDA belongs to the Ydjc family and is the only known enzyme which selectively catalyze the deacetylation of acetylglucosamine under mild conditions in vitro.It is meaningful to elucidate the specific selectivity of CmCBDA for GlcNAc which is useful for the development of high-quality biocatalysts for the production of GlcN.In this paper,we solved the complex structure of CmCBDA with metal Mn ion and reaction product acetic acid molecule.The experimental results show that CmCBDA exhibits a dimeric form in solution,consistent with an asymmetric unit containing two molecules of CmCBDA.The overall structure of CmCBDA is in the shape of an a-helix wrapped ?-sheet,and the pocket of the enzyme is located in the loop of the ?-sheet and its surroundings.Further analysis of the structure revealed that Asp22,His72 and His 143 were involved in chelation of Mn ion,while Asp21,His230 and Arg253 were involved in the binding to acetate molecule.By comparing the other two Ydjc family proteins in PDB,we hypothesized that the selectivity of CmCBDA for GlcNAc may be related to its narrow and closed binding pocket.Our work laid a foundation for further elucidation of the catalytic mechanism,substrate selectivity and subsequent enzyme engineering of CmCBDA.