recombinant pentaketide chromone synthasedoes not produce chalcone from 4-coumaroyl-CoA, but instead efficiently accepts malonyl-CoA as a sole substrate to carry out iterative condensations of five molecules of malonyl-CoA, producing a pentaketide, 5,7-dihydroxy-2-methylchromone. Recombinant pentaketide chromone synthaseaccepts acetyl-CoA, resulting from decarboxylation of malonyl-CoA, as a starter substrate
the plant-specific type III polyketide synthase that produces 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA. The aromatic pentaketide is a biosynthetic precursor of the anti-asthmatic furochromones kehellin and visnagin
pentaketide chromone synthase is a plant-specific type III polyketide synthase that belongs to the chalcone synthase superfamily of type III polyketide synthases
pentaketide chromone synthase is a plant-specific type III polyketide synthase that belongs to the chalcone synthase superfamily of type III polyketide synthases and grouped with other non-chalcone forming enzymes
active site structure of wild-type and mutant enzymes, overview. Amino acid residue at position 197 in the active site governs the chain length of the polyketide. Leucine at position 256 in the active site for both oktaketide synthase and pentaketide chromone synthase influences the substrate preference for malonyl-CoA as a starting unit, while a glycine residue located in the same position and found in the catalytic pocket of chalcone synthase can possibly compel the enzyme to readily accept 4-coumaroyl-CoA as a starting unit
pentaketide chromone synthase residue Met207 is fairly flexible and adopts at least two distinct conformations, active site structure, catalytic residues of pentaketide chromone synthase are V351, M207, and L266, overview. Residues Cys143, Thr204, Met207, Leu266, and Val351 influence the catalytic activity and differentiate the enzyme from chalcone synthase, EC 2.3.1.74. The pentaketide chromone synthase crystal structure reveals neither a hydrogen-bond network, nor any additional catalytic Cys residues that appears to be crucial for the enzyme reaction
the critical active-site residue 197, and the catalytic triad, Cys164, His303, and Asn336, are conserved in type III polyketide synthases, while the residues lining the active-site are exchanged to M197, L256, V338 (numbering in Madia sativa CHS). Active-site architecture of pentaketide chromone synthase compared to other type III polyketide synthases, crystal structure and hmology modeling, overview
purified detagged recombinant pentaketide chromone synthase, hanging drop vapour diffusion method, mixing of 500 nl of 10 mg/ml protein in 20 mM HEPES-NaOH, pH 7.0, containing 100 mM NaCl, and 2 mM DTT, with 500 nl reservoir solution containing 100 mM Tris-HCl, pH 8.5, 14% w/v PEG 8000, and 350 mM KF, 20°C, 2 days, X-ray diffraction structure determination and analysis at 1.6 A resolution
site-directed mutagenesis, the total cavity volume of the F80A/Y82A/M207G triple mutant is 4fold larger than that of the wild-type pentaketide chromone synthase. The mutant not only catalyzes the iterative condensation of nine molecules of malonyl-CoA, to produce anovel nonaketide naphthopyrone, but also alters the mechanism of the cyclization to produce the angular naphthopyrone with a fused tricyclic ring system
site-directed mutagenesis, the mutant enzyme, in contrast to the wild-type, efficiently catalyzes the successive condensation of eight molecules of malonyl-CoA to produce SEK4 and SEK4b. The pentaketide-forming pentaketide chromone synthase is thus functionally transformed into an octaketide-producing enzyme by the single amino-acid substitution, the mutant performs a C-10/C-15 aldol-type cyclization reaction, the double mutant is almost functionally identical to the single mutant M207G
site-directed mutagenesis, the mutant enzyme, in contrast to the wild-type, efficiently catalyzes the successive condensation of eight molecules of malonyl-CoA to produce SEK4 and SEK4b. The pentaketide-forming pentaketide chromone synthase is thus functionally transformed into an octaketide-producing enzyme by the single amino-acid substitution, the mutant performs a C-10/C-15 aldol-type cyclization reaction, the double mutant is almost functionally identical to the single mutant M207G
site-directed mutagenesis, the mutant enzyme, in contrast to the wild-type, efficiently catalyzes the successive condensation of eight molecules of malonyl-CoA to produce SEK4 and SEK4b. The pentaketide-forming pentaketide chromone synthase is thus functionally transformed into an octaketide-producing enzyme by the single amino-acid substitution, the mutant performs a C-10/C-15 aldol-type cyclization reaction, the double mutant is almost functionally identical to the single mutant M207G
site-directed mutagenesis, the mutant enzyme, in contrast to the wild-type, efficiently catalyzes the successive condensation of eight molecules of malonyl-CoA to produce SEK4 and SEK4b. The pentaketide-forming pentaketide chromone synthase is thus functionally transformed into an octaketide-producing enzyme by the single amino-acid substitution, the mutant performs a C-10/C-15 aldol-type cyclization reaction, the double mutant is almost functionally identical to the single mutant M207G
site-directed mutagenesis, the mutant enzyme, in contrast to the wild-type, efficiently catalyzes the successive condensation of eight molecules of malonyl-CoA to produce SEK4 and SEK4b. The pentaketide-forming pentaketide chromone synthase is thus functionally transformed into an octaketide-producing enzyme by the single amino-acid substitution, the mutant performs a C-10/C-15 aldol-type cyclization reaction, the double mutant is almost functionally identical to the single mutant M207G
oktaketide synthase, EC 2.3.1.-, and pentaketide chromone synthase, EC 2.3.1.216, are not functionally interconvertible by the single amino acid switch at residue 207
site-directed mutagenesis, the mutant enzyme efficiently catalyzes the successive condensation of eight molecules of malonyl-CoA to produce 2,7-dihydroxy-5-[(4-hydroxy-2-oxo-2H-pyran-6-yl)methyl]-2-methyl-2,3-dihydro-4H-chromen-4-one and 2,7-dihydroxy-5-[(4-hydroxy-2-oxo-2H-pyran-6-yl)methyl]-5-methyl-2,3-dihydro-4H-chromen-4-one, i.e. SEK4 and SEK4b. The pentaketide-forming pentaketide chromone synthase is thus functionally transformed into an octaketide-producing enzyme by the single amino-acid substitution
site-directed mutagenesis, the mutant enzyme, in contrast to the wild-type, efficiently catalyzes the successive condensation of eight molecules of malonyl-CoA to produce 2,7-dihydroxy-5-[(4-hydroxy-2-oxo-2H-pyran-6-yl)methyl]-2-methyl-2,3-dihydro-4H-chromen-4-one and 2,7-dihydroxy-5-[(4-hydroxy-2-oxo-2H-pyran-6-yl)methyl]-5-methyl-2,3-dihydro-4H-chromen-4-one, i.e. SEK4 and SEK4b. The pentaketide-forming pentaketide chromone synthase is thus functionally transformed into an octaketide-producing enzyme by the single amino-acid substitution, the mutant performs a C-10/C-15 aldol-type cyclization reaction
site-directed mutagenesis, the pentaketide chromone synthase M207G mutant no longer produces the pentaketide chromone but instead efficiently catalyzes sequential condensations of eight molecules of malonyl-CoA to produce a 1:4 mixture of the octaketides SEK4/SEK4b. The pentaketide-producing pentaketide chromone synthaseis thus transformed into an octaketide synthase by the single-amino acid replacement
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally GST-tagged pentaketide chromone synthase from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, removal of the GST-tag, anion exchange chromatography, and gel filtration to homogeneity
use of pentaketide chromone synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds