Novel dual-function type III polyketide synthase from Hypericum polyphyllum

Benzophenone synthase (BPS) and biphenyl synthase (BIS) are benzoic acid-specific type III polyketide synthases (PKSs). They use the rare starter substrate benzoyl-CoA and three molecules of malonyl-CoA as extender substrate to form the same linear tetraketide intermediate [Fig. 1], BPS catalyzes intramolecular C6→C1 Claisen condensation to form 2,4,6-trihydroxybenzophenone [1], whereas BIS catalyzes intramolecular C2→C7 aldol condensation to form 3,5-dihydroxybiphenyl [2]. Polyprenylated benzophenones with interesting pharmacological features, such as cytotoxic [3] and antibacterial [4] activities, are specialized metabolites in some Hypericum sp. Biphenyls and related dibenzofurans are phytoalexins of the Rosaceae subtribe Malinae [5].

A type III polyketide synthase was cloned from H. polyphyllum, which forms 2,4,6-trihydroxybenzophenone and 3,5-dihydroxybiphenyl in in vitro assay. Therefore, it is a good candidate for investigating the two different cyclization mechanisms by undertaking mutations in the active center of the enzyme.

The incubation temperature influenced the product ratio. The enzyme produced only 3,5-dihydroxybiphenyl until 30 °C. Above, 2,4,6-trihydroxybenzophenone formation started and both products were observed in equal amounts at 45 °C. Under optimum conditions (45 °C, 300 mM KH₂PO₄, pH 7,5), V _max value was 300 nkat/mg and Km values were 4.5 µM for benzoyl-CoA and 26.4 µM for malonyl-CoA.

Two single amino acid mutations in the active center changed the product ratio

(2,4,6-trihydroxy-benzophenone:

3,5-dihydroxybiphenyl) to 1: 0.5 and 1: 0.2, respectively. The double mutant produced only 2,4,6-trihydroxybenzophenone.

Thus, the PKS is a promising candidate for crystallization which will provide in-depth information about the active site structure and the switch in the cyclization modes.

Fig. 1 Reactions of benzo-phenone synthase (BPS) and biphenyl synthase (BIS) [2].

• References

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