Literature summary for 2.1.1.281 extracted from

Zou, X.W.; Liu, Y.C.; Hsu, N.S.; Huang, C.J.; Lyu, S.Y.; Chan, H.C.; Chang, C.Y.; Yeh, H.W.; Lin, K.H.; Wu, C.J.; Tsai, M.D.; Li, T.L.
Structure and mechanism of a nonhaem-iron SAM-dependent C-methyltransferase and its engineering to a hydratase and an O-methyltransferase (2014), Acta Crystallogr. Sect. D, 70, 1549-1560.

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Cloned(Commentary)

Cloned (Comment)	Organism
gene mppJ from the mannopeptimycin-biosynthetic gene cluster, DNA and amino acid sequence determination and analysis, recombinant expression of N-terminally His6-tagged enzyme	Streptomyces hygroscopicus

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His6-tagged enzyme, in apoform or in complex with S-adenosyl-L-methionine and phenylpyruvate, hanging drop vapour diffusion technique, mixing of 0.001 ml of 24 mg/ml protein in 50 mM HEPES, pH 7.5, 100 mM CaCl2, with 0.001 ml of reservoir solution containing 16% PEG 3350, 200 mM NaI, 20°C, 10 days, X-ray diffraction structure determination and analysis at 2.0-2.5 A resolution, molecular replacement. From the collected crystal diffraction data four additional structures of MppJ in complex with phenlpyruvate, 4-hydroxyphenylpyruvate, S-adenosyl-L-methionine and phenylpyruvate or S-adenosyl-L-homocysteine and methylphenylpyruvate, although crystals of the apo form remain unobtainable	Streptomyces hygroscopicus

Crystallization (Comment)

Organism

purified recombinant His6-tagged enzyme, in apoform or in complex with S-adenosyl-L-methionine and phenylpyruvate, hanging drop vapour diffusion technique, mixing of 0.001 ml of 24 mg/ml protein in 50 mM HEPES, pH 7.5, 100 mM CaCl2, with 0.001 ml of reservoir solution containing 16% PEG 3350, 200 mM NaI, 20°C, 10 days, X-ray diffraction structure determination and analysis at 2.0-2.5 A resolution, molecular replacement. From the collected crystal diffraction data four additional structures of MppJ in complex with phenlpyruvate, 4-hydroxyphenylpyruvate, S-adenosyl-L-methionine and phenylpyruvate or S-adenosyl-L-homocysteine and methylphenylpyruvate, although crystals of the apo form remain unobtainable

Streptomyces hygroscopicus

Protein Variants

Protein Variants	Comment	Organism
C319A	site-directed mutagenesis, the mutant shows 39% reduced activity compared to the wild-type enzyme	Streptomyces hygroscopicus
D244E	site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme	Streptomyces hygroscopicus
D244L	site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme	Streptomyces hygroscopicus
R127L/D244E	site-directed mutagenesis, the mutant enzyme shows altered conformation and substrate binding structure compared to the wild-type enzyme	Streptomyces hygroscopicus
W99F	the mutant shows altered stereochemistry in the reaction compared to the wild-type enzyme	Streptomyces hygroscopicus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	required, binding structure, overview	Streptomyces hygroscopicus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
38000	-	2 * 38000, recombinant His6-tagged enzyme, SDS-PAGE	Streptomyces hygroscopicus
70200	-	recombinant His-tagged enzyme, gel filtration	Streptomyces hygroscopicus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
S-adenosyl-L-methionine + 3-phenylpyruvate	Streptomyces hygroscopicus	phenylpyruvate binding structure analysis	S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate	-	?
S-adenosyl-L-methionine + 3-phenylpyruvate	Streptomyces hygroscopicus NRRL3085	phenylpyruvate binding structure analysis	S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate	-	?

Organism

Organism	UniProt	Comment	Textmining
Streptomyces hygroscopicus	Q643C8	gene mppJ	-
Streptomyces hygroscopicus NRRL3085	Q643C8	gene mppJ	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged enzyme by nickel affinity chromatography, ion-exchange chromatography, and ultrafiltration	Streptomyces hygroscopicus

Reaction

Reaction	Comment	Organism	Reaction ID
S-adenosyl-L-methionine + 3-phenylpyruvate = S-adenosyl-L-homocysteine + (3S)-2-oxo-3-phenylbutanoate	the enzyme methylates the benzylic C atom of phenylpyruvate to give 3-methylpyruvate, reaction mechanism, structure-function analysis, overview	Streptomyces hygroscopicus	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	the enzyme has no nonheme oxygenase activity	Streptomyces hygroscopicus	?	-	?
additional information	the enzyme has no nonheme oxygenase activity	Streptomyces hygroscopicus NRRL3085	?	-	?
S-adenosyl-L-methionine + 3-phenylpyruvate	phenylpyruvate binding structure analysis	Streptomyces hygroscopicus	S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate	-	?
S-adenosyl-L-methionine + 3-phenylpyruvate	Arg127 is electrostatically associated with phenylpyruvate, binding structure analysis, enzyme MppJ is an R-configuration-specific methyltransferase, ratios of 5:1 and 1:1 for (2S,3R)-betaMePhe versus (2S,3S)-betaMePhe	Streptomyces hygroscopicus	S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate	-	?
S-adenosyl-L-methionine + 3-phenylpyruvate	phenylpyruvate binding structure analysis	Streptomyces hygroscopicus NRRL3085	S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate	-	?
S-adenosyl-L-methionine + 3-phenylpyruvate	Arg127 is electrostatically associated with phenylpyruvate, binding structure analysis, enzyme MppJ is an R-configuration-specific methyltransferase, ratios of 5:1 and 1:1 for (2S,3R)-betaMePhe versus (2S,3S)-betaMePhe	Streptomyces hygroscopicus NRRL3085	S-adenosyl-L-homocysteine + (3R)-2-oxo-3-phenylbutanoate	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * 38000, recombinant His6-tagged enzyme, SDS-PAGE	Streptomyces hygroscopicus

Synonyms

Synonyms	Comment	Organism
MppJ	-	Streptomyces hygroscopicus

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	-	Streptomyces hygroscopicus

General Information

General Information	Comment	Organism
evolution	enzyme MppJ is structurally related to the MT protein family	Streptomyces hygroscopicus
additional information	the enzyme is composed of two domains: a dimerization domain (DD) and a methyltransfer domain (MT). The substrate-binding pocket is situated at the occlusion of the DD and MT domains, in which the iron centre is solvent-accessible	Streptomyces hygroscopicus