Understanding Programming of Fungal Iterative Polyketide Synthases: The Biochemical Basis for Regioselectivity by the Methyltransferase Domain in the Lovastatin Megasynthase

Abstract

Highly reducing polyketide synthases (HR-PKSs) from fungi synthesize complex natural products using a single set of domains in a highly programmed, iterative fashion. The most enigmatic feature of HR-PKSs is how tailoring domains function selectively during different iterations of chain elongation to afford structural diversity. Using the lovastatin nonaketide synthase LovB as a model system and a variety of acyl substrates, we characterized the substrate specificity of the LovB methyltransferase (MT) domain. We showed that, while the MT domain displays methylation activity toward different β-ketoacyl groups, it is exceptionally selective toward its naturally programmed β-keto-dienyltetraketide substrate with respect to both chain length and functionalization. Accompanying characterization of the ketoreductase (KR) domain displays broader substrate specificity toward different β-ketoacyl groups. Our studies indicate that selective modifications by tailoring domains, such as the MTs, are achieved by higher kinetic efficiency on a particular substrate relative to the rate of transformation by other competing domains.

Figure 1

The programed steps of LovB in the synthesis of dihydromonacolin L (DML). (A) The catalytic steps by LovB; LovB is a HR-PKS and LovC is the dissociated enoylreductase; and (B) the tetraketide modification steps shown in detail highlighting the timing of the MT domain. Domain abbreviations: ketosynthase (KS); malonyl-CoA:ACP acyltransferase (MAT); α-methyltransferase (MT), β-ketoreductase (KR), dehydratase (DH), α–β enoylreductase (ER), acyl carrier protein (ACP) and NRPS-like Condensation (CON).

Figure 2

Full kinetic analysis of LovB MT domain towards different β-ketoacyl-SNAC substrates. 2 is the natural tetraketide substrate based on DML structure. 8 is the on-pathway triketide substrate of LovB. No reaction towards diketide 7 or pentaketide 11 was observed.

Figure 3

KR and MT competition assays using model and natural tri- and tetraketide substrates. (A) Quantification of product distribution of model substrates 9 and 10. (B) and (C) Product distributions of natural substrates 8 and 2, respectively. Shown are the extract ion chromatograms of different products as indicated.