Substrate specificity of the polyketide synthase 6-methylsalicylic acid synthase multienzyme complex isolated from penicillium patulum and investigation of its malonyl-coa decarboxylase activity

Campuzano, Iain David Grant (1998) Substrate specificity of the polyketide synthase 6-methylsalicylic acid synthase multienzyme complex isolated from penicillium patulum and investigation of its malonyl-coa decarboxylase activity. University of Southampton, Doctoral Thesis.

Abstract

The multienzyme complex, 6-methylsalicylic acid synthase catalyses the formation of the simple polyketide 6-methylsalicylic acid, from one molecule of acetyl-CoA, three molecules of malonyl-CoA and one equivalent of the reducing cofactor NADPH. In the absence of NADPH triacetic acid, lactone is exclusively synthesised.

Upon incubation of 6-methylsalicylic acid synthase with alternative starter substrate units in the presence and absence of NADPH, the corresponding 6-alkylsalicyclic acid analogues and triketide lactone analogues were biosynthesised respectively. There was an inverse relationship between the carbon chain length of the starter unit and the level of incorporation into the final product. The 6-alkylsalicyclic acid analogues and triketide lactone analogues were purified and characterised by reverse phase high pressure liquid chromatography and electrospray mass spectrometry.

Using succinyl-CoA transferase, purified from porcine heart, [2-¹³C]malonyl-CoA was biosynthesised. Making use of a linked assay, ¹³C-labelled 6-methylsalicyclic acid and ¹³C-labelled triacetic acid lactone were produced in the absence of externally added acetyl-CoA. By collision induced dissociation mass spectroscopy analysis, it was determined that 6-methylsalicyclic acid synthase possesses a native malonyl-CoA decarboxylase activity.

I also report the first ever mass measurement of a fully functional polyketide synthase subunit using nanospray time-of-flight mass spectrometry and electrospray quadrupole mass spectrometry.

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