Structural And Funtional Analysis Of CMP Hydroxymethylase And N-Glycosidase Invovled In Mildiomycin Biosynthesis

5-Hydroxymethylcytosine(5hmC),also known as the 'sixth base',was first discovered in T4 phage DNA in the mid-20th.Since this time,5hmC have been found in mammalian DNA,and played an important role in gene expression and regulation,cell differentiation and embryonic development.In addition,the 5hmC moiety was also found in some bacteria-derived natural products,such as mildiomycinMildiomycin(MIL)is a peptidyl nucleoside antibiotic isolated from the culture filtrate of Streptomyces rimofaciens with strong activity against powdery mildew disease of plants and has the advantages of high efficiency,low toxicity,broad spectrum,environmental friendliness and the pathogen has not shown resistant to it.Cytidine monophosphate(CMP)hydroxymethylase MilA and CMP N-glycosidase MilB catalyzed the formation of hydroxymethyl cytosine to initiate the MIL biosynthesisMilA is the first enzyme of thymidylate synthetase and pyrimidine hydroxymethylase superfamily preferring CMP over deoxycytidylate(dCMP)as supported by a catalytic efficiency(k,at/KM)for CMP 5-fold higher than for dCMP and catalyzes the synthesis of 5-hydroxymethyl CMP(5hmCMP).We thus determined the crystal structures of MilA and its complexes with various substrates including CMP,dCMP and 5hmCMP.Comparing these structures to those of dCMP hydroxymethylase(CH)from T4 phage and TS from Escherichia coli revealed that a common serine(Ser)in the active site of CH and TS is replaced by an alanine(Ala176)in MilA.In this superfamily,the residue(Ala/Ser)of this site plays an important role in the recognition of ribosyl-substrate/deoxyribosyl-substrate.On the basis of the structural information,the mutation of MilA A176S was constructed and in vitro reactions confirmed that this mutant could reverse the substrate preference of Mil A from CMP to dCMP.In terms of the reaction mechanism,after the common step in which the methylene of 5,10-methylenetetrahydrofolate(5,10-CH2-THF)is transferred to the 5-carbon of the substrate,TS transfers a hydride form the intermediate(THFA)to the exocyclic methylene,forming the product of thymidine monophosphate(dTMP).However,different from TS,both MilA and CH utilize a water molecule from the solvent to catalyze the hydration of the exocyclic methylene of the substrate and produce hydroxymethylated products.In this study,based on the crystal structures of MilA and its complexes with substrates,structural comparisons with TS/CH,and sequences alignment,the functions of relevant amino acids were explored to clarify the structural basis of the differences in catalytic specificity.In addition,the crystal structure of MilA shows that it has an additional domain consisting of over 90 amino acids in the carboxyl terminal.Construction of the C-terminal truncation mutants of MilA,MilA 1-235 and MilA 1-249,shows that both of them are insoluble.We proposed that this extra C-terminal domain may be related to the correct folding of Mil A.The hmCMP produced by MilA is the substrate for the CMP N-glycosidase Mi1B which catalyzed the second step reaction in the biosynthesis of mildiomycin.MilB belongs to the family of nucleoside deoxyribose transferase(NDT)and catalyzes the hydrolysis of the N-glycosidic bond of 5hmCMP to produce 5hmC.MilB is the first enzyme of this family to specifically recognize hmCMP.In this study,the kinetic parameters of MilB on substrates of CMP and 5hmCMP were determined,indicating that the KM of MilB for CMP is?27-fold higher than that for hmCMP.We determined the crystal structures of MilB and its catalytically inactive E103 A mutant in complex with hmCMP.Remarkably,upon hmCMP binding,the guanidinium group of Arg23 was bent toward hmCMP to recognize its 5-hydroxymethyl group,inducing semi?closure of the cage-like pocket.Mutagenesis studies of Arg23 and bioinformatics analysis demonstrate that the positively charged Arg/Lys at this site is critical for the specific recognition of the 5-hydroxymethyl group of hmCMP.In summary,this work focuses on the enzymatic and structural studies of MilA and MilB,which are responsible for 5hmC synthesis in the initial steps of mildiomycin biosynthesis.In this study,the crystal structures of MilA,MilA-CMP,MilA-dCMP,MilA-5hmCMP and MilB,MilB E103A-5hmCMP were determined,and the relationship between their structures and functions were further discussed.Remarkably,these studies on substrate preference of MilA and MilB,have given a better understanding of the structures and functional evolution of their superfamily and laid solid foundation for biosynthesis of engineered MIL analogs in the future.