A Plant Type III Polyketide Synthase that Produces Pentaketide Chromone

Abstract

A novel plant-specific type III polyketide synthase (PKS) that catalyzes formation of a pentaketide chromone, 5,7-dihydroxy-2-methylchromone, from five molecules of malonyl-CoA, was cloned and sequenced from aloe (Aloe arborescens). Site-directed mutagenesis revealed that Met207 (corresponding to Thr197 in CHS) determines the polyketide chain length and the product specificity of the enzyme; remarkably, replacement of a single amino acid residue, Met207, with Gly yielded a mutant enzyme that efficiently produces aromatic octaketides, SEK4 and SEK4b, the products of the minimal PKS for actinorhodin (act from Streptomyces coelicolor), from eight molecules of malonyl-CoA. This provided new insights into the catalytic functions and specificities of the CHS-superfamily type III PKS enzymes.

Type III polyketide synthases in natural product biosynthesis

“…Additionally, OKS has broad substrate specificity, as hexanoylCoA has been found to be a better substrate in the formation of two novel compounds, C 18 heptaketide phloroglucinol and C 16 hexaketide resorcinol (15). Similar to OKS, the enzyme pentaketide chromone synthase (PCS) can take malonyl-CoA as both the starter and extender units, catalyzing sequential decarboxylative condensation of five molecules of malonyl-CoA to produce 5,7-dihydroxy-2-methylchromone in A. arborescens (16). PCS and OKS share 91% of their amino acid sequences but catalyze different reactions.…”

“…However, the mutant PKS efficiently produced an unnatural pentaketide, 2,7-dihydroxy-5-methylchromone that is a regioisomer of 5,7-dihydroxy-2-methylchromone produced by PCS (13). Similarly, the PCS M207G mutant produced the octaketides SEK4/SEK4b instead of the pentaketide chromone (16). These results suggested that there must be additional subtle structural differences of the active-site architecture between the two enzymes and the single residue 207 played a crucial role in controlling the polyketide chain length and product specificity (15).…”

Cloning and Functional Analysis of a Novel Aldo-Keto Reductase from Aloe arborescens

“…For the PKR assay, 17) the reaction mixture contained 54 mM of cinnamoyl-CoA (or cinnamoyldiketide-NAC), 108 mM of malonyl-CoA, 1 mM NADPH, and 5 mg of plant polyketide synthases (A. arborescence pentaketide chromone synthase, 18) octaketide synthase, 19) or Scutellaria baicalensis CHS 17) ), and 60 mg of A. arborescens AKR4C12 (or Glycyrrhiza echinata PKR) in a final volume of 500 ml of 100 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. Incubations were carried out at 30°C for 20 min to overnight, and the products were analyzed by the reversephase HPLC as described before.…”

“…16,17) To further study the plant PKR enzymes, we carried out cloning of PKRs from a medicinal plant Aloe arborescens (Liliaceae) that produces considerable amount of the "reduced" polyketides including anthrones and anthraquinones. 18,19) Here we report a PKR homolog obtained by a combination of RT-PCR using degenerate primers based on the conserved sequences of plant PKRs (chalcone reductases) and cDNA library screening by oligonucleotide hybridization. Sequence, function, and homology modeling analyses revealed that this is a novel AKR superfamily enzyme that belongs to subfamily 4C12.…”

Synthesis of unnatural alkaloid scaffolds by exploiting plant polyketide synthase

“…Interestingly, type III PKSs exhibit remarkable substrate tolerance, accepting a series of nonphysiological substrate analogues to produce chemically and structurally distinct unnatural novel polyketides (4)(5)(6)(7)(8)(9)(10). Furthermore, recent structure-based engineering of functionally divergent type III PKSs has significantly expanded the catalytic repertoire of the enzymes and the product diversity (11)(12)(13)(14)(15)(16)(17). Due to their remarkable substrate promiscuity and catalytic potential, the structurally and mechanistically simple type III PKS enzymes represent an excellent platform for the further development of unnatural novel biocatalysts with unprecedented catalytic functions (18).…”