Characterization And Enzymatic Engineering Of CMP Hydroxymethylase MilA

CMP hydroxymethylase (MilA) involved in the biosynthesis of mildiomycin, is the only confirmed enzyme with substrate bias to ribonucleotide monophosphate (CMP). However, in its superfamily, both thymidylate synthase (TS) and dCMP hydroxymethylase (CH) prefer to catalyze deoxyribonucleotide monophosphate as substrates(dUMP and dCMP respvetively). The unrevealed mechanism of their discrepancy on ribose/deoxyribose specificity is of interest in the research on necleotide metabolism.To confirm MilA's preference to CMP, catalysis efficiency of MilA toward CMP and dCMP were characterized, and the efficiency toward CMP is 8.5 times higher than that toward dCMP. In vitro assay of MilA fed with H218O showed that 18O was incorporated into the hydroxymethyl-CMP, which proved similar catalytic mechanism between MilA and dCMP hydroxymethylase.Bioinformatic prediction of secondary and three-dimensional structure of MilA, along with multiple alignment with its homologs in the database, revealed that despite as limited as below 30% amino acid identity, secondary and three-dimensional structures of MilA is very similar to that of CH and TS. Moreover, in the substrate binding cavity of MilA, Lys133 and Ala176 were predicted to govern MilA's preference to ribose monophosphate, due to their relatively short side chains of the key amino acid residues.Based on the hypothesis, we designed, constructed and purified three single mutants (MilA K133R,A176S,A176T) and two double mutants (A176S/K133R,A176T/K133R) with relatively longer side chain on residue 133 and 176. In vitro reaction of these mutants revealed that MilA A176T and MilA A176T/K133R could not use CMP any more, while maintaining their ability to catalyze the hydroxymethylation of dCMP.In conclusion, compared to CH, MilA's substrate specificity toward ribose monophosphate is determined by Ala176. And single mutation of A176T can lead to loss of activity on CMP.