The Product Specificities of Maize Terpene Synthases TPS4 and TPS10 Are Determined both by Active Site Amino Acids and Residues Adjacent to the Active Site

Abstract

Terpene synthases make up a large family of enzymes that convert prenyl diphosphates into an enormous variety of terpene skeletons. Due to their electrophilic reaction mechanism-which involves the formation of carbocations followed by hydride shifts and skeletal rearrangements-terpene synthases often produce complex mixtures of products. In the present study, we investigate amino acids that determine the product specificities of the maize terpene synthases TPS4 and TPS10. The enzymes showed 57% amino acid similarity and produced different mixtures of sesquiterpenes. Sequence comparisons and structure modeling revealed that out of the 43 amino acids forming the active site cavity, 17 differed between TPS4 and TPS10. While combined mutation of these 17 residues in TPS4 resulted in an enzyme with a product specificity similar to TPS10, the additional mutation of two amino acids next to the active site led to a nearly complete conversion of TPS4 into TPS10. These data demonstrate that the different product specificities of TPS4 and TPS10 are determined not only by amino acids forming the active site cavity, but also by neighboring residues that influence the conformation of active site amino acids.

Keywords: 7-epi-sesquithujene; Zea mays; sesquiterpene synthase; site-directed mutagenesis; terpene synthase reaction mechanism; α-bergamotene; β-bisabolene; β-farnesene.

Figure 1

Comparison of the reaction mechanisms catalyzed by TPS4 and TPS10. The major TPS4 products 7-epi-sesquithujene and β-bisabolene are marked by green boxes, while the major TPS10 products (E)-β-farnesene and (E)-α-bergamotene are marked by red boxes. Green and red arrows indicate main steps in the reaction sequences of TPS4 and TPS10, respectively.

Figure 2

Comparison of the deduced amino acid sequences of TPS4 and TPS10. Amino acids identical in both proteins are marked by black boxes. Amino acids situated at the surface of the active site cavity are highlighted by arrowheads. The white diamonds indicate the two mutated residues outside the active site cavity. Helices J and K and the J-K loop are indicated.

Figure 3

Site-directed mutagenesis of 17 active site amino acids differing between TPS4 and TPS10. The effects of single mutations, s1–s17 (A) and combined mutations, c1–c14 (B), on product specificity were studied. Genes were heterologously expressed in Escherichia coli and partially purified enzymes were incubated with (E,E)-farnesyl diphosphate (FPP) in the presence of the cofactor Mg²⁺. Total ion chromatograms of gas chromatography-mass spectrometry (GC-MS) analyses of the terpene products are shown. The sesquiterpene olefins were identified by mass spectrometry and comparison of mass spectra and retention times to those of authentic standards. 1, 7-epi-sesquithujene; 2, (E)-α-bergamotene; 3, (E)-β-farnesene; 4, β-bisabolene. The expression of soluble terpene synthase (TPS) enzymes was tested by western blot analysis of E. coli raw protein extracts using a TPS4-specific antibody. Data for single mutants (C) and combinatorial mutants (D) are shown.

Figure 4

Active site amino acids and residues adjacent to them determine the different product specificities of TPS4 and TPS10. Product profiles of the mutants TPS4 I408V + A409G + T410A + A412Q + L413V (A) and TPS4-C17 R442K + I411F (B) and wild-type TPS10 (C) are shown. Genes were heterologously expressed in Escherichia coli and partially purified enzymes were incubated with (E,E)-farnesyl diphosphate (FPP) in the presence of the cofactor Mg²⁺. Total ion chromatograms of gas chromatography-mass spectrometry (GC-MS) analyses of the terpene products are shown. The sesquiterpene olefins were identified by mass spectrometry and comparison of mass spectra and retention times to those of authentic standards. 1, 7-epi-sesquithujene; 2, (E)-α-bergamotene; 3, (E)-β-farnesene; 4, β-bisabolene; 5, β-sesquiphellandrene; 6, zingiberene.

Figure 5

Model of the three-dimensional structure of the mutant TPS4-c17. The homology-based model was created using the crystal structure of 5-epi-aristolochene synthase from tobacco as template and allowed the identification of arginine 442 (A) and isoleucine 411 (B) as non-active site residues potentially influencing the product specificity. The pictures illustrate different views into the active site cavity. Residues lining the cavity are shown in blue and adjacent residues are shown in red. The helices G1 and G2 are labeled.