Information on EC 2.1.2.B2 - cytidylate 5-hydroxymethyltransferase

Substrates: MilA uses solvent water as the hydroxyl group donor like cytidylate hydroxymethylase. Thr102 of MilA is a potential critical amino acid anchoring one molecule of water for hydroxylation. Thr102 of MilA is predicted as potential critical amino acid anchoring one molecule of water for hydroxylation
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Substrates: MilA catalyzes the conversion of CMP into 5-hydroxymethyl-CMP with an efficiency (kcat/KM) of 5fold faster than for deoxycytidylate
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additional information

Substrates: MilA catalyzes the conversion of CMP into 5-hydroxymethyl-CMP with an efficiency (kcat/KM) of 5fold faster than for deoxycytidylate
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additional information

Substrates: 5-hydroxymethylcytosine is present in a nucleoside antibiotic mildiomycin and its formation is governed by a cytidylate hydroxymethylase MilA
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show all columns Go to Natural Substrate Search

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NATURAL SUBSTRATE

NATURAL PRODUCT

REACTION DIAGRAM

ORGANISM

UNIPROT

LITERATURE

COMMENTARY

REVERSIBILITY
r=reversible
ir=irreversible
?=not specified

5,10-methylenetetrahydrofolate + H2O + CMP

tetrahydrofolate + 5-hydroxymethyl-CMP

5,10-methylenetetrahydrofolate + H2O + cytidylate

tetrahydrofolate + 5-hydroxymethylcytidylate

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5,10-methylenetetrahydrofolate + H2O + CMP

tetrahydrofolate + 5-hydroxymethyl-CMP

Substrates: -
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5,10-methylenetetrahydrofolate + H2O + CMP

tetrahydrofolate + 5-hydroxymethyl-CMP

Substrates: -
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5,10-methylenetetrahydrofolate + H2O + cytidylate

tetrahydrofolate + 5-hydroxymethylcytidylate

Substrates: -
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5,10-methylenetetrahydrofolate + H2O + cytidylate

tetrahydrofolate + 5-hydroxymethylcytidylate

Substrates: -
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additional information

Substrates: 5-hydroxymethylcytosine is present in a nucleoside antibiotic mildiomycin and its formation is governed by a cytidylate hydroxymethylase MilA
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additional information

Substrates: 5-hydroxymethylcytosine is present in a nucleoside antibiotic mildiomycin and its formation is governed by a cytidylate hydroxymethylase MilA
Products: -

show all columns Go to Cofactor Search

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COFACTOR

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

5,10-methylenetetrahydrofolate

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KM VALUE [mM]

SUBSTRATE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

0.0719

CMP

at pH 7.5 and 37°C

0.245

dCMP

at pH 7.5 and 37°C

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TURNOVER NUMBER [1/s]

SUBSTRATE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

0.047

CMP

at pH 7.5 and 37°C

0.032

dCMP

at pH 7.5 and 37°C

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kcat/KM VALUE [1/mMs^-1]

SUBSTRATE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

0.65

CMP

at pH 7.5 and 37°C

0.13

dCMP

at pH 7.5 and 37°C

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pH OPTIMUM

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

7.5

assay at

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TEMPERATURE OPTIMUM

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

assay at

show all columns Go to Organism Search

only manually annotated data include textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks include all textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks + FRENDAco-occurrence of enzyme names and organism names (less precise)

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ORGANISM

COMMENTARY

LITERATURE

UNIPROT

SEQUENCE DB

SOURCE

3 entries

brenda

UniProt

brenda

gene milA

brenda

brenda

gene milA

brenda

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GENERAL INFORMATION

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

evolution

metabolism

4 entries

physiological function

additional information

evolution

since MilA have similar mechanism as CH in hydroxyl group formation, it may belong to the superfamily of thymidylate synthase and cytidylate hydroxymethylase

evolution

since MilA have similar mechanism as CH in hydroxyl group formation, it may belong to the superfamily of thymidylate synthase and cytidylate hydroxymethylase

metabolism

mildiomycin formation pathway catalyzed by MilA and MilB, overview

metabolism

the enzyme is involved in mildiomycin biosynthesis

metabolism

mildiomycin formation pathway catalyzed by MilA and MilB, overview

metabolism

the enzyme is involved in mildiomycin biosynthesis

physiological function

several nucleotide-derived nucleoside antibiotics feature modified bases such as hydroxymethyl pyrimidine in polyoxin, and hydroxymethyl cytosine in mildiomycin. The CMP hydroxymethylase named MilA in the biosynthetic pathway of nucleoside mildiomycin in Streptoverticillum rimofaciens ZJU5119 can convert CMP in vitro in the presence of tetrahydrofolate to 5-hydroxymethyl-CMP that is immediately hydrolyzed into free 5-hydroxymethylcytosine by MilB

physiological function

additional information

six homologous ORFs originally annotated as putative thymidylate synthase are more likely to be CMP hydroxymethylase

additional information

six homologous ORFs originally annotated as putative thymidylate synthase are more likely to be CMP hydroxymethylase

Go to AA Sequence and Transmembrane Helices SearchGo to Localization Prediction

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UNIPROT

ENTRY NAME

ORGANISM

NO. OF AA

NO. OF TRANSM. HELICES

MOLECULAR WEIGHT[Da]

SOURCE

SEQUENCE

LOCALIZATION PREDICTION

The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).

B4Y380 pBLAST

B4Y380_9ACTN

334

37379

TrEMBL

Show Sequence

Secretory Pathway (Reliability: 2)

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SUBUNIT

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

homodimer

homodimer

homodimer

Go to Crystallization (commentary) Search

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CRYSTALLIZATION (Commentary)

ORGANISM

UNIPROT

LITERATURE

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

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

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PROTEIN VARIANTS

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

A176S/K133R

3 entries

A176S/K133R

mutation results in a reversal of substrate preference from CMP to dCMP

A176S/K133R

the mutation results in completely eliminated catalytic efficiency toward CMP while its efficiency to dCMP is slightly affected

A176S/K133R

the mutation results in completely eliminated catalytic efficiency toward CMP while its efficiency to dCMP is slightly affected

Go to Purification (commentary) Search

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PURIFICATION (Commentary)

ORGANISM

UNIPROT

LITERATURE

Ni-NTA column chromatography and Superdex S200 gel filtration

recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography

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CLONED (Commentary)

ORGANISM

UNIPROT

LITERATURE

expressed in Escherichia coli BL21(DE3) cells

gene milA, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)

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REF.

AUTHORS

TITLE

JOURNAL

VOL.

PAGES

YEAR

ORGANISM (UNIPROT)

PUBMED ID

SOURCE

Chen, C.; Gao, T.; Zhao, G.; Deng, Z.; Hu, S.; Xu, H.; He, X.

Evidence from 18O feeding studies for hydroxyl group donor in the reaction catalyzed by cytidylate hydroxymethylase MilA

Chin. Sci. Bull.

864-868

2013

Streptomyces rimofaciens, Streptomyces rimofaciens ZJU5119

brenda