Evidence for catalytic cysteine-histidine dyad in chalcone synthase

Chalcone and stilbene synthases (CHS and STS) catalyze condensation reactio ns of p-coumaroyl-CoA and three C-2-units from malonyl-CoA, but catalyze di fferent cyclization reactions to produce naringenin chalcone and resveratro l, respectively. Condensing activities of wild-type CHS and STS as well as STS-C60S mutant were inhibited by iodoacetamide (Idm) and diethyl pyrophosp hate (DPC), DPC also inhibited malonyl-CoA decarboxylation activity of wild -type and C164S mutants of CHS and STS, Meanwhile, Idm treatment enhanced ( two- to fourfold) malonyl decarboxylase activity of wild-type enzymes and S TS-C60S, whereas this priming effect was not observed with C164S mutants of CHS and STS, indicating that the cysteine residue being modified by Idm is the catalytic Cys164 of CHS and STS. DPC inhibition of decarboxylation act ivity of wild-type CHS was pH-independent in the range of pH 5.8 to 7.8; ho wever, its inhibitory effect on CHS-C164S increased as pH increased from 6. 2 to 7.4 with a midpoint of 6.4. Based on the 3-D structure of CHS and the observed shift in microscopic pK(a), it was concluded that the histidine re sidue being modified by DPC in CHS is Likely the catalytic His303 and that His303 forms an ionic pair (catalytic dyad) with Cys164 in wild-type CHS. i n addition, our results showed that Cys60 in STS is not essential for the a ctivity and only a single cysteine (Cys164) participates in the catalysis a s in CHS. (C) 2000 Academic Press.