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PMID:16144421

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Citation

Abe, I, Oguro, S, Utsumi, Y, Sano, Y and Noguchi, H (2005) Engineered biosynthesis of plant polyketides: chain length control in an octaketide-producing plant type III polyketide synthase. J. Am. Chem. Soc. 127:12709-16

Abstract

The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) produces a variety of plant secondary metabolites with remarkable structural diversity and biological activities (e.g., chalcones, stilbenes, benzophenones, acrydones, phloroglucinols, resorcinols, pyrones, and chromones). Here we describe an octaketide-producing novel plant-specific type III PKS from aloe (Aloe arborescens) sharing 50-60% amino acid sequence identity with other plant CHS-superfamily enzymes. A recombinant enzyme expressed in Escherichia coli catalyzed seven successive decarboxylative condensations of malonyl-CoA to yield aromatic octaketides SEK4 and SEK4b, the longest polyketides known to be synthesized by the structurally simple type III PKS. Surprisingly, site-directed mutagenesis revealed that a single residue Gly207 (corresponding to the CHS's active site Thr197) determines the polyketide chain length and product specificity. Small-to-large substitutions (G207A, G207T, G207M, G207L, G207F, and G207W) resulted in loss of the octaketide-forming activity and concomitant formation of shorter chain length polyketides (from triketide to heptaketide) including a pentaketide chromone, 2,7-dihydroxy-5-methylchromone, and a hexaketide pyrone, 6-(2,4-dihydroxy-6-methylphenyl)-4-hydroxy-2-pyrone, depending on the size of the side chain. Notably, the functional diversity of the type III PKS was shown to evolve from simple steric modulation of the chemically inert single residue lining the active-site cavity accompanied by conservation of the Cys-His-Asn catalytic triad. This provided novel strategies for the engineered biosynthesis of pharmaceutically important plant polyketides.

Links

PubMed Online version:10.1021/ja053945v

Keywords

Acyltransferases/chemistry; Acyltransferases/metabolism; Aloe/chemistry; Aloe/enzymology; Amino Acid Sequence; Macrolides/chemical synthesis; Macrolides/chemistry; Macrolides/metabolism; Molecular Sequence Data; Molecular Structure; Plant Proteins/chemistry; Plant Proteins/metabolism; Plant Roots/chemistry; Protein Biosynthesis; Time Factors

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ALOAR:OKS

enables

GO:0016747: acyltransferase activity, transferring groups other than amino-acyl groups

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ALOAR:OKS

involved_in

GO:0009813: flavonoid biosynthetic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ALOAR:OKS

enables

GO:0042803: protein homodimerization activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ALOAR:Q3L7F5

GO:0030639: polyketide biosynthetic process

ECO:0000314:

P

Figure 1D and Figure 5A shows that OKS uses 8 malonyl- CoA as a sole substrate to yield a 1:4 mixture of octaketides SEK4 and SEK4b.

complete
CACAO 4084

ALOAR:Q3L7F5

involved_in

GO:0009813: flavonoid biosynthetic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

ALOAR:Q3L7F5

enables

GO:0016747: transferase activity, transferring acyl groups other than amino-acyl groups

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ALOAR:Q3L7F5

enables

GO:0042803: protein homodimerization activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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