Crystal Structure And Substrate Selection Mechanism Of Adenylation Domain Of D-alanylase Involved In The Biosynthesis Of Ansatrienins

Ansatrienins are 21-membered macrolactams of the ansamycin family,which bear characteristic N-acyl-D-alanyl modification at the C-11 position.The N-acyl-D-alanyl side chain is very important for the biological activity of ansatrienins.Previous studies have confirmed that the side chain is sequentially catalyzed by a unique D-alanylase AstC and acyltransferase AstF1.D-alanylase AstC is a single-module non-ribosomal peptide synthase(NRPS)composed of "A-T-TE" domains.The adenylation domain(A domain)is responsible for the activation of D-alanine,and the thioesterase domain catalyzes the formation of ester bonds.Maytansine is another important subclass of ansamycins.It has an N-acyl-L-alanyl side chain at the C-3 position,which is obviously similar to the N-acyl-D-alanyl side chain of ansatrienins.We speculate that it is also biosynthesized by a similar mechanism.AstC exhibit weak D-alanylation activity at C-3 position of maytansinoid in vivo,which provides some support for this hypothesis.At the same time,this result also points out the direction for the production of L-alanyl-maytansinol,an important anti-tumor drug precursor,through synthetic biology approaches.In order to elucidate the structural characteristics of adenylation domain of D-alanylase and its recognition mechanism for D-Ala,we have studied the A domain of AstC and its homologs AnsC,MycC,and SY.The progress as follows:1)The basic physicochemical properties of A domain of D-alanylase were established and the truncated protein expression system was optimized to achieve high-level expression and purification of the target protein.2)The complex structures of SY-A protein with D-Ala-SA or D-Phe-AMP substrate were analyzed by crystallization conditions search and optimization,diffraction data collection,and processing.The complex structure contains two asymmetric units of protein molecules with basically the same three-dimensional structure,and both of them form AMP binding pockets and amino acid binding pockets,which are located at the cleft between Acore and Asub subdomains 3)Through structure superposition and sequence comparison of homologs,the key amino acid residues in the substrate binding pocket were located.The in vitro enzyme activity experiments of the corresponding mutants confirmed that the highly conserved residues played an important role in the adenylation catalytic reaction,and when the amino acid substrate steric hindrance was large,the substrate might take non-classical binding towards the other pocket.A domain of Dalanylase had an obvious preference for D-Ala and tolerance for Gly and slightly larger sterically hindered amino acids.4)SY-A was modified by mutagenesis to accommodate substrate L-Ala.Combined with crystal structure and computer-aided simulation,the mutants with improved activity and selectivity to L-Ala were obtained,which provided directions and ideas for subsequent transformation,though did not achieve exclusive selectivity to L-Ala.In this thesis,we elucidated the mechanism of acyl donor amino acid substrate recognition by heterologous expression and structural biology studies of D-alanylase of ansatrienins,and attempted to improve the catalytic activity by mutagenesis,which laid a solid foundation for the further rational/semi-rational engineering to create maytansinoid L-alanylase.