Abstract
Modified pyrimidine monophosphates such as methyl dCMP (mdCMP), hydroxymethyl dUMP (hmdUMP) and hmdCMP in some phages are synthesized by a large group of enzymes termed as thymidylate synthases (TS). Thymidylate is a nucleotide required for DNA synthesis and thus TS is an important drug target. In the biosynthetic pathway of the nucleoside fungicide mildiomycin isolated from Streptomyces rimofaciens ZJU5119, a cytidylate (CMP) hydroxymethylase, MilA, catalyzes the conversion of CMP into 5′-hydroxymethyl CMP (hmCMP) with an efficiency (kcat/KM) of 5-fold faster than for deoxycytidylate (dCMP). MilA is thus the first enzyme of the TS superfamily preferring CMP to dCMP. Here, we determined the crystal structures of MilA and its complexes with various substrates including CMP, dCMP and hmCMP. Comparing these structures to those of dCMP hydroxymethylase (CH) from T4 phage and TS from Escherichia coli revealed that two residues in the active site of CH and TS, a serine and an arginine, are respectively replaced by an alanine and a lysine, Ala176 and Lys133, in MilA. Mutation of A176S/K133R of MilA resulted in a reversal of substrate preference from CMP to dCMP. This is the first study reporting the evolution of the conserved TS in substrate selection from DNA metabolism to secondary nucleoside biosynthesis.
Highlights
Since TS is responsible for the production of dTMP, one of the building blocks for DNA synthesis, it has been extensively studied as a target for cancer chemotherapy[18]
We demonstrated that MilA has a substrate preference for CMP over dCMP, and offers an opportunity to investigate the mechanism by which conserved TS evolves the preference for ribosyl over 2′-deoxyribosyl groups
Liquid chromatography-mass spectroscopy (LC-MS) detected the ion corresponding to the product hydroxymethyl dCMP (hmdCMP) ([M +H]+ mass = 338, retention time Rt = 16.5 min), its UV absorption peak was covered by that of the tetrahydrofolate (THFA) (Rt = 16.8 min) (Fig. S1A)
Summary
Equal concentrations of CMP and dCMP were added in the same reaction system with MilA to compete with each other, and hmdCMP and THFA were completely separated using an optimized elution condition in high-performance liquid chromatography (HPLC) analysis. The kinetic parameters for MilA were determined with either CMP or dCMP as its substrate (Table 1, Fig. S2). The kcat/KM for hmCMP was 39.2 mM−1 min−1, 5-fold higher than that for hmdCMP (kcat/KM = 7.84 mM−1 min−1, Table 1). Prompted by this observation, we performed the structural comparison of MilA with CH and other TS members to identify the amino acids of MilA critical for its substrate preference for ribosyl cytidylate. There is no obvious difference between the structures of CMP-bound MilA and apo MilA, with the root-mean-square deviation (RMSD)
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