The Acyl Transferase Activity Of An Epimerase MarH And The Biosynthesis Of Sparsomycin Scarffold

Most of the small molecular natural compounds with diverse bioactivities are secondary metabolites,including polyketides,nonribosomal peptides and their hybrids,etc.The biosyntheses of natural products follow rigorous and universal principle,also implicate novel enzymatic reactions.Several enzymes with unexpected activities were studied in this thesis.Maremycins,a series of diketopiperazines containing indole moiety with anti-tumor effects,are produced by marine-derived Streptomyces sp.B9173.The cupin protein MarH was previously characterized as an epimerase involved in the?2S,3S?-?-methyltryptophan formation in the maremycin biosynthesis.When MarH was overexpressed in E.coli BL21?DE3?plysS,we accidentally discovered that the strain heterogeneously expressing MarH could acetylate chloramphenicol?Cml?faster than the control strain.The in vitro assays showed that purified MarH could catalyze3-O-acetylation of Cml using acetyl-CoA as an acetyl donor.Then the acetyl group transfered to 1-OH spontaneously,and 3-OH was acetylated by MarH again to generate 1,3-O-diacetyl Cml.It indicates that MarH is a multifunctional enzyme with both epimerase and acetyltransferase activities.Studies of the reaction kinetics indicated that StnK3,ACPL₆197 and Cwoe₄835,three homologues of MarH,had comparative catalytic efficiency to canonical CATs.And the in vivo assays showed that marH could exist as a Cml resistance gene.By systematic site-directed mutagenesis,a similar catalytic model of MarH was proposed to that of canonical CATs.H64 in cupin motif 1 was revealed as a potential catalytic base that deprotonates the acyl acceptor substrate.Nucleophilic attack at the carbonyl carbon of the acyl donor then gives the acylation product.MarH showed a strong selectivity on acyl acceptors and a relative flexibility on acyl donors.It only acetylated the R-configuration acceptors,while it could recognize several acyl-CoAs with simple structures.Furthermore,MarH can catalyze N-acylation of deacylated Cml analogue thereby initiating 3-O-acylation.It indicates that MarH catalyzes the acetylation of-NH₂ as well as-OH,and prefers the-NH₂ group over the-OH group.MarH and homologous proteins consist of only about 130 amino acid residues,but can simultaneously catalyze two distinct reactions?isomerization reactions and acyl transfer reaction?,which is very innovative and rare in nature.Our study will greatly expand our understanding of the catalytic mechanism of the enzyme.Sparsomycin?SPS?with broad-spectrum antimicrobial and antitumor activities is produced by S.sparsogenes NRRL2940.SPS is composed of6-methyl-uracil acrylic acid?UAA?and oxo-methylthiomethyl-cysteinol?O-MTM-cysteinol?linked by an amide bond.According to previous isotope feeding results,the UAA monomer is derived from tryptophan and the O-MTM-cysteinol part is derived from cysteine and methionine.The structure of SPS implies that its gene cluster should include two NRPS modules.A novel cloning strategy was set up by cloning SPS resistance gene as a probe and the biosynthetic gene cluster of SPS was obtained.Bioinformatics analyses showed that there were two genes encoding unexpected three NRPS modules in the gene cluster.All these three NRPS modules were proved essential for SPS biosynthesis through gene knock-out experiments.Then substrate specificities of these three A domains were tested respectively in order to figure out the three NRPS modules'real roles in SPS biosynthesis.The three A-PCP didomains were heterologously overexpressed and purified.The in vitro adenylation activities of these three didomains towards different substrates were detected.The results indicated that A1 domain and A3 domain were responsible for UAA's and MTM-L-Cys's activation respectively,while A2 domain did not show any specific activity towards the tested compounds.With the cooperation of Wenjun Zhang's group,we further demonstrated that the MTM-L-Cys was firstly transfered from PCP3to PCP2 domain by SpsC,a type II TE,was then converted to MTM-D-Cys by C/E domain.And the SPS scaffold structure was finally formed through the condensation of MTM-D-Cys and UAA catalyzed by C domain.The study on the biosynthesis of SPS scaffold provides a theoretical basis for systematic exploration and genetic engineering of SPS biosynthesis.Since A1 domain could also activate other aromatic acrylic acids,non-natural precursors guided biosynthesis of SPS analogues was therefore carried out.These attempts have laid the foundation for the development of new antitumor drugs.