Literature summary for 2.1.2.B2 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 .

Search for more literature for 2.1.2.B2

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli BL21(DE3) cells	Streptomyces rimofaciens

Crystallization (Commentary)

Crystallization (Comment)	Organism
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
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

Protein Variants	Comment	Organism
A176S/K133R	mutation results in a reversal of substrate preference from CMP to dCMP	Streptomyces rimofaciens
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]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Streptomyces rimofaciens	-	-	-
Streptomyces rimofaciens	B4Y380	-	-
Streptomyces rimofaciens ZJU5119	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
Ni-NTA column chromatography and Superdex S200 gel filtration	Streptomyces rimofaciens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
5,10-methylenetetrahydrofolate + H2O + CMP	-	Streptomyces rimofaciens	tetrahydrofolate + 5-hydroxymethyl-CMP	-	?
5,10-methylenetetrahydrofolate + H2O + CMP	CMP is the strongly preferred substrate over dCMP	Streptomyces rimofaciens	tetrahydrofolate + 5-hydroxymethyl-CMP	-	?
5,10-methylenetetrahydrofolate + H2O + CMP	-	Streptomyces rimofaciens ZJU5119	tetrahydrofolate + 5-hydroxymethyl-CMP	-	?
5,10-methylenetetrahydrofolate + H2O + CMP	CMP is the strongly preferred substrate over dCMP	Streptomyces rimofaciens ZJU5119	tetrahydrofolate + 5-hydroxymethyl-CMP	-	?
5,10-methylenetetrahydrofolate + H2O + cytidylate	-	Streptomyces rimofaciens	tetrahydrofolate + 5-hydroxymethylcytidylate	-	?
5,10-methylenetetrahydrofolate + H2O + dCMP	-	Streptomyces rimofaciens	tetrahydrofolate + 5-hydroxymethyl-dCMP	-	?
5,10-methylenetetrahydrofolate + H2O + dCMP	-	Streptomyces rimofaciens ZJU5119	tetrahydrofolate + 5-hydroxymethyl-dCMP	-	?
5,10-methylenetetrahydrofolate + H2O + deoxycytidylate	-	Streptomyces rimofaciens	tetrahydrofolate + 5-hydroxymethyldeoxycytidylate	-	?
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

Subunits	Comment	Organism
homodimer	-	Streptomyces rimofaciens

Synonyms

Synonyms	Comment	Organism
CMP hydroxymethylase	-	Streptomyces rimofaciens
MilA	-	Streptomyces rimofaciens
thymidylate synthase	-	Streptomyces rimofaciens

Turnover Number [1/s]

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

General Information

General Information	Comment	Organism
metabolism	the enzyme is involved in mildiomycin biosynthesis	Streptomyces rimofaciens

kcat/KM [mM/s]

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

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Release 2023.1 (January 2023)