Identification of amino acid residues important in the cyclization reactions of chalcone and stilbene synthases

Abstract

Chalcone synthase (CHS) and stilbene synthase (STS) catalyse condensation reactions of p-coumaroyl-CoA and three C(2) units from malonyl-CoA up to a common tetraketide intermediate but then catalyse different cyclization reactions to produce naringenin chalcone and resveratrol respectively. On the basis of sequence alignment with other condensing enzymes including 3-ketoacyl-(acyl carrier protein) synthases of polyketide and fatty-acid synthases, site-directed mutagenesis was performed on the active-site G(372)FGPG loops in CHS and STS. The CHS-P375G mutant showed a 6-fold decrease in overall condensing activity with selectively increased production of p-coumaroyltriacetic acid lactone (CTAL, the derailment product of the tetraketide intermediate). Meanwhile, resveratrol production by STS-P(375)G strongly decreased to give various products in the order CTAL> resveratrol approximately bisnoryangonin>naringenin. As a result, naringenin production (cross-reaction) by STS-P(375)G was close to 30% of resveratrol production. Both G(374)L mutants of CHS and STS showed no condensing activity with residual malonyl-CoA decarboxylase activity. These results suggested that the G(372)FGPG loop in CHS and STS contribute to a determination of the outcome during cyclization reactions by serving as a part of the active-site scaffold on which the stereochemistry of cyclization is performed. These observations provide the first biochemical indication that cyclization reactions are modulated by active-site geometry. The implications for the evolutionary relationship of these enzymes are also discussed.

Inferring Hypotheses on Functional Relationships of Genes: Analysis of the Arabidopsis thaliana Subtilase Gene Family

“…It is very likely that a few amino acid changes could alter the substrate specificity of a protease. A striking example of the consequences of a single amino acid change on the properties of an enzyme is provided by the stilbene synthases [50]. …”

In silicio expression analysis of PKS genes isolated from Cannabis sativa L.

“…The catalytic triad (Cys157, His297, and Ans330), the “gatekeeper” phenylalanines (Phe208 and Phe259), and Met130, which ties one catalytic site to the other one on the homodimeric complex, as well as Gly250, which determines the elongation cavity volume of the active site, are strictly preserved when compared to CHS2 from alfalfa (Ferrer et al , 1999; Jez et al , 2000a; Jez et al , 2001). The GFGPG loop, which is important for the cyclization reactions in CHS/STS-type PKSs (Suh et al , 2000), is also preserved on our PKSs. In the starter substrate-binding pocket, the amino acid residues Ser126 and Ser332 are also preserved as on alfalfa CHS2, but Glu185, Thr187 and Thr190 are replaced by an Asp, a Met and a Leu, respectively.…”

“…The models suggest small differences in the local reorientation of the residues that shape the active site of the cannabis PKSs. The substrate and product specificity of the enzyme reaction can be affected by the steric modulation of the active-site architecture (Ferrer et al , 1999; Jez et al , 2000a,b, 2001; Suh et al , 2000). It can be inferred from these models that cannabis PKSs could have differences in substrate specificity or in catalytic efficiency ( k

Figure 2 The relative orientation of the side chains of the active site residues from M. sativa CHS with the 3D models of C. sativa PKS.

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Phylogeny and Expression Analyses Reveal Important Roles for Plant PKS III Family during the Conquest of Land by Plants and Angiosperm Diversification

“…This is consistent with the fact that changes in only a few amino acids were enough to change the qualitative enzyme functions from 2-PS or ACS to CHS (Jez et al, 2000; Abe et al, 2007). Although current experimental efforts concerning CHS and STS functions only quantitatively exchanged their substrate preferences (Schröder and Schröder, 1992; Suh et al, 2000). Attempts to exchange functions between CHS and STS enzymes requires more investigation.…”

“…In addition, a single change of His to Glu at position 166 alters the substrate preference of AhSTS from p -coumaroyl-CoA to cinnamoyl-CoA (Schröder and Schröder, 1992). But current efforts in CHS and STS conversion only partially alter the catalytic reactions through active sites or their geometry (Tropf et al, 1995; Suh et al, 2000). More evidence is needed for these CHS and STS enzymes, even with the help of the crystalline structures of STS (Shomura et al, 2005).…”