title: Structural basis of the substrate preference towards CMP for a thymidylate synthase MilA involved in mildiomycin biosynthesis.
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.
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 China.
authors.forename: Gong
authors.initials: G
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xiong
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 China.
authors.forename: Tuling
authors.initials: T
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 China.
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 China.
authors.forename: Geng
authors.initials: G
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 China.
authors.forename: Xinyi
authors.initials: X
authors.lastname: He
mesh_terms: D000409:Alanine / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D018360:Crystallography, X-Ray; D003568:Cytidine Monophosphate / Q000378:metabolism*; D003596:Cytosine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D003841:Deoxycytidine / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012266:Ribose / Q000737:chemistry; D013302:Streptomyces / Q000737:chemistry*; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003841:Deoxycytidine; D012266:Ribose; D003596:Cytosine; D013940:Thymidylate Synthase; D003568:Cytidine Monophosphate; D008239:Lysine; D000409:Alanine; C017539:mildiomycin