Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates.
Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase.
Watanabe
K
K
Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. kenji@chem.agr.hokudai. ac.jp
Oikawa
H
H
Yagi
K
K
Ohashi
S
S
Mie
T
T
Ichihara
A
A
Honma
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001565:Benzoates / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry; D015723:Gene Library; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D010071:Oxaloacetates / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011753:Pyrones / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D013997:Time Factors
D001565:Benzoates; D018076:DNA, Complementary; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D010071:Oxaloacetates; D010455:Peptides; D011753:Pyrones; C408144:macrophomate synthase; D019289:Pyruvic Acid