Structural and functional studies on 6-methylsalicylic acid synthase from Penicillium patulum and holo-acy1 carrier protein synthase from Escherichia coli

The type I polyketide synthase, 6-methylsalicylic acid synthase, is a multifunctional enzyme that catalyses the formation of 6-methylsalicylic acid from one starter unit of acetyl-CoA, three extender units of malonyl-CoA and one equivalent of the reducing cofactor NADPH. In the absence of NADPH, a triketide intermediate is released from the enzyme as triacetic acid lactone.Using succinyl-CoA transferase, purified from porcine heart, [2-13C]-malonyl-CoA was biosynthesised and in a linked assay, [13C]-labelled 6-methylsalicylci acid and [13C]-labelled triacetic acid lactone were produced in the absence of externally added acetyl-CoA. By collision induced dissociation mass spectrometric analysis, it was determined that 6-methylsalicylic acid synthase possesses a malonyl-CoA decarboxylase activity. Similar methodology was also used to show that in the absence of NADPH, 6-methylsalicyclic acid synthase is able to synthesise small amounts of orsellinic acid as well as triacetic acid lactone. It is proposed that in the absence of NADPH, the triketide intermediate is a poor substrate for the β-ketosynthase but that 1-2% can react with malonyl-CoA to give orsellinic acid.Fluoroacetyl-CoA, chloroacetyl-CoA, and bromoacetyl-CoA were synthesised and purified and their effect/action with 6-methylsalicylic acid synthase was investigated. All were shown to inactivate the enzyme although none were incorporated into halo-products. The interaction between 6-methylsalicylic acid synthase and several N-acetylcysteamine intermediates was also investigated. The enzyme appears to prefer to synthesise its own intermediates rather than accept externally added substrate analogues.