Evolution History Of PhlG Homologues And Characterization Of Phloretin Hydrolase MbPhlG In Mycobacterium Abscessus

The structure and function of PhlG in Pseudomonas fluorescens Pf-5 have been determined, it catalyzes DAPG and generates MAPG. However, even in high PhlG sequence similarity species, gene context of PhlG possesses much difference, which indicates those PhlG homologues have various functions.We analyze the evolutionary history of PhlG in different species and performe a positive selection analysis on the PhlG of Mycobacterium abscessus 103 and Pseudomonas fluorescens 2P24(MaPhlG and PfPhlG) at the same time we claasify PhlG and its homologues into 6 different clusters. Besides, we identify the MaPhlG in M.abscessus 103 is the result of horizontal gene transfer.We identify the function of MaPhlG、PfPhlG and MtPhlG(from Mycobacterium tuberculosis).MtPhlG is a type of synthase, which can react on phloretin and generate new compounds, it breaks C-C bond and form new C-C bond. PfPhlG is one kind of DAPG hydrolase with high substrate-specificity. The FCA-HHI mutant of PfPhlG can react on DAPG and MAPG, which indicates PfPhlG may be under the selection of DAPG during evolution. MaPhlG is a relative ―broad-spectrum‖ enzyme and it can catalyze phloretin,MAPG and DAPG.Compared with MaPhlG,when we mutated H160 and I162(in MaPhlG)to F and A,we find the mutants have higher DAPG hydrolase activity and lower hydrolysis of MAPG,indicating that FCA are essential to DAPG hydrolysis so as HHI are to MAPG. Q266 L have low activity on MAPG and phloretin.It almost lose activity on DAPG, illustrating that MaPhlG evolves to a higher activity enzyme. We found PG, cinnamic acid and phloretic acid could inhibit MaPhlG functioning on phloretin with higer Ki, indicating the low activity of phloretin is not the result of product inhibition.