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Literature summary extracted from

  • Zhao, G.; Chen, C.; Xiong, W.; Gao, T.; Deng, Z.; Wu, G.; He, X.
    Structural basis of the substrate preference towards CMP for a thymidylate synthase MilA involved in mildiomycin biosynthesis (2016), Sci. Rep., 6, 39675 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.2.B2 expressed in Escherichia coli BL21(DE3) cells Streptomyces rimofaciens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.1.2.B2 enzyme complexed with various substrates including CMP, dCMP and 5'-hydroxymethyl-CMP, hanging drop vapor diffusion method, using 15% (w/v) polyethylene glycol 3350, 0.08 M lithium sulfate monohydrate and 0.1 M Tris-HCl, pH 8.5 Streptomyces rimofaciens
2.1.2.B2 structures of MilA and its complexes with substrates including CMP, dCMP and hydroxymethyl-CMP. Two residues in the active site of dCMP hydroxymethylase from T4 phage and thymidylate synthase from Escherichia coli, a serine and an arginine, are respectively replaced by an alanine and a lysine, Ala176 and Lys133, in MilA Streptomyces rimofaciens

Protein Variants

EC Number Protein Variants Comment Organism
2.1.2.B2 A176S/K133R mutation results in a reversal of substrate preference from CMP to dCMP Streptomyces rimofaciens
2.1.2.B2 A176S/K133R the mutation results in completely eliminated catalytic efficiency toward CMP while its efficiency to dCMP is slightly affected Streptomyces rimofaciens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.2.B2 0.0719
-
CMP at pH 7.5 and 37°C Streptomyces rimofaciens
2.1.2.B2 0.245
-
dCMP at pH 7.5 and 37°C Streptomyces rimofaciens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + CMP Streptomyces rimofaciens
-
tetrahydrofolate + 5-hydroxymethyl-CMP
-
?
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + CMP Streptomyces rimofaciens ZJU5119
-
tetrahydrofolate + 5-hydroxymethyl-CMP
-
?

Organism

EC Number Organism UniProt Comment Textmining
2.1.2.B2 Streptomyces rimofaciens
-
-
-
2.1.2.B2 Streptomyces rimofaciens B4Y380
-
-
2.1.2.B2 Streptomyces rimofaciens ZJU5119
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.2.B2 Ni-NTA column chromatography and Superdex S200 gel filtration Streptomyces rimofaciens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + CMP
-
Streptomyces rimofaciens tetrahydrofolate + 5-hydroxymethyl-CMP
-
?
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + CMP CMP is the strongly preferred substrate over dCMP Streptomyces rimofaciens tetrahydrofolate + 5-hydroxymethyl-CMP
-
?
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + CMP
-
Streptomyces rimofaciens ZJU5119 tetrahydrofolate + 5-hydroxymethyl-CMP
-
?
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + CMP CMP is the strongly preferred substrate over dCMP Streptomyces rimofaciens ZJU5119 tetrahydrofolate + 5-hydroxymethyl-CMP
-
?
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + cytidylate
-
Streptomyces rimofaciens tetrahydrofolate + 5-hydroxymethylcytidylate
-
?
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + dCMP
-
Streptomyces rimofaciens tetrahydrofolate + 5-hydroxymethyl-dCMP
-
?
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + dCMP
-
Streptomyces rimofaciens ZJU5119 tetrahydrofolate + 5-hydroxymethyl-dCMP
-
?
2.1.2.B2 5,10-methylenetetrahydrofolate + H2O + deoxycytidylate
-
Streptomyces rimofaciens tetrahydrofolate + 5-hydroxymethyldeoxycytidylate
-
?
2.1.2.B2 additional information MilA catalyzes the conversion of CMP into 5-hydroxymethyl-CMP with an efficiency (kcat/KM) of 5fold faster than for deoxycytidylate Streptomyces rimofaciens ?
-
-

Subunits

EC Number Subunits Comment Organism
2.1.2.B2 homodimer
-
Streptomyces rimofaciens

Synonyms

EC Number Synonyms Comment Organism
2.1.2.B2 CMP hydroxymethylase
-
Streptomyces rimofaciens
2.1.2.B2 MilA
-
Streptomyces rimofaciens
2.1.2.B2 thymidylate synthase
-
Streptomyces rimofaciens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.1.2.B2 0.032
-
dCMP at pH 7.5 and 37°C Streptomyces rimofaciens
2.1.2.B2 0.047
-
CMP at pH 7.5 and 37°C Streptomyces rimofaciens

General Information

EC Number General Information Comment Organism
2.1.2.B2 metabolism the enzyme is involved in mildiomycin biosynthesis Streptomyces rimofaciens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.1.2.B2 0.13
-
dCMP at pH 7.5 and 37°C Streptomyces rimofaciens
2.1.2.B2 0.65
-
CMP at pH 7.5 and 37°C Streptomyces rimofaciens