INVESTIGATION OF THE MECHANISM AND STERIC COURSE OF THE REACTION CATALYZED BY 6-METHYLSALICYLIC ACID SYNTHASE FROM PENICILLIUM-PATULUM USING (R)-[1-C-13 2-H-2]MALONATE AND (S)-[1-C-13 2-H-2]MALONATE

Chiral malonyl-CoA derivatives, enzymically synthesized from (R)- and (S)-[1-C-13;2-H-2]malonates using succinyl-CoA transferase, were incorporated into 6-methylsalicylic acid with homogeneous 6-methylsalicylic acid synthase isolated from Penicillium patulum. Analysis of the 6-methylsalicylic acid formed established that the hydrogen atoms at the 3- and 5-positions are derived from opposite absolute configurations in malonyl-CoA. When acetoacetyl-CoA was used as the starter molecule, a single hydrogen atom is incorporated from the chiral malonates into the 3-position of the 6-methylsalicylic acid. Mass spectrometric analysis of the 6-methylsalicylic acid indicates that this hydrogen atom originates from H(Re) of malonyl-CoA or H(Si) in the polyketide intermediate. It is thus concluded that the hydrogen atom at the 5-position of 6-methylsalicylic acid originates from H(Si) of malonyl-CoA or H(Re) in the polyketide intermediate. During the reaction the enzyme also catalyzes the stereospecific exchange of hydrogen atoms in the polyketide intermediates. The impliciations of the stereochemical information from these experiments are discussed in relation to the mechanism of the 6-methylsalicylic acid synthase reaction.