A single change of histidine to glutamine alters the substrate preference of a stilbene synthase.
Stilbene and chalcone synthases are related polyketide synthases which use the same substrates but form different products. The environment of the condensing active site cysteine is highly conserved, except for the positions -2 and -3. All chalcone synthases contain Gln-Gln and prefer 4-coumaroyl-CoA as starter CoA ester, while the two known stilbene synthases contain Gln-His or His-Gln (preference phenylpropionyl-CoA and 4-coumaroyl-CoA, respectively). We investigated whether the presence and/or position of the histidine influences the substrate preference and the product specificity (stilbene or chalcone). The two amino acid motifs in the chalcone synthase from Pinus sylvestris (Gln-Gln) and in the stilbene synthases from P. sylvestris (Gln-His) and Arachis hypogaea (His-Gln) were changed by site-directed mutagenesis into all sequence combinations as found in the natural enzymes. Assays with the mutant proteins showed that the histidine does not determine the product specificity. With the chalcone and the stilbene synthase from P. sylvestris, any sequence deviation reduced the activity without marked effects on the substrate preference. The stilbene synthase from A. hypogaea was different. The change from His-Gln to Gln-His abolished enzyme activity almost completely with all three substrates. The change to Gln-Gln selectively reduced the activity with 4-coumaroyl-CoA, and the kinetic analysis indicated a slight increase in Km and a 3-fold reduction of Vmax, when compared with the parent enzyme. This converted the enzyme from a resveratrol-forming into a dihydropinosylvin-forming stilbene synthase.
Schröder
G
G
Institut für Biologie II, Universität Freiburg, Federal Republic of Germany.
Schröder
J
J
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D005973:Glutamine; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005973:Glutamine; D006639:Histidine; D000217:Acyltransferases; C022674:stilbene synthase