Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning, functional expression, and site‐directed mutagenesis of two polyketide synthases

Abstract

SummaryBenzophenone derivatives, such as polyprenylated benzoylphloroglucinols and xanthones, are biologically active secondary metabolites. The formation of their C 13 skeleton is catalyzed by benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of Hypericum androsaemum. BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53±63% amino acid sequence identity. Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept activated cinnamic acids as starter molecules. In contrast, recombinant chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a phylogenetic tree, the two PKSs occurred in different clusters. One cluster was formed by CHSs including the one from H. androsaemum. BPS grouped together with the PKSs that functionally differ from CHS. Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoylCoA over 4-coumaroyl-CoA.

Alkylresorcinol Synthases Expressed in Sorghum bicolor Root Hairs Play an Essential Role in the Biosynthesis of the Allelopathic Benzoquinone Sorgoleone

“…The 100% relative activity corresponds to 81.0 pkat mg À1 for ARS1 and 60.1 pkat mg À1 for ARS2, defined as pmol of alkylresorcinol formed/s per mg of protein. (Table 1), indicated k cat and k cat /K m values in range with those obtained for other type III plant PKSs using preferred substrates (e.g., Jez et al, 2000;Liu et al, 2003;Abe et al, 2005b;Katsuyama et al, 2009;Taura et al, 2009). A single derailment product was observed for ARS1 and ARS2 in enzyme assays using saturated acyl-CoA substrates from hexanoyl-CoA (C6) to myristoyl-CoA (C14), which was not detectable for substrates longer than C14.…”

Metabolic Engineering of the Phenylpropanoid Pathway in Saccharomyces cerevisiae

“…CHS is a type III polyketide synthase that has broad substrate specificity (18,32,34). CHS from H. androsaemum condenses either p-coumaroyl-CoA or cinnamoyl-CoA with three molecules of malonyl-CoA to produce the corresponding chalcones (25). The identification of phloretin and 2Ј,4Ј,6Ј-trihydroxydihydrochalcone suggests that dihydrocinnamoyl-CoA and dihydro-p-coumaroyl-CoA are further catalyzed by CHS.…”

Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

“…In cell cultures of Hypericum androsaemum (St. John's wort family of plants), BPS catalyzes the decarboxylative condensation of three units of malonyl-CoA with one unit of benzoyl-CoA to form a tetraketide intermediate, which undergoes an intramolecular C6 to C1 Claisen condensation to give 2,4,6-trihydroxybenzophenone (25, Scheme 7) [51]. Intriguingly, H. androsaemum CHS showed 60% identity to BPS, and was able to utilize benzoyl-CoA as a starter with low activity to form benzophenone.…”