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Pak. J. Bot., 48(1): 231-239, 2016.

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MUTATIONAL, PHYLOGENY AND EVOLUTION ANALYSES OF SALVIA COPALYL DIPHOSPHATE SYNTHASE

DA CHENG HAO^1*, RAMESHA B. THIMMAPPA² AND PEI GEN XIAO³

Abstract: The cyclization of geranylgeranyl diphosphate (GGPP) is catalyzed by copalyl diphosphate synthase (CPS), a class II diterpene synthase (diTPS), to form copalyl diphosphate (CPP), which is an essential substrate of a variety of diterpenes in secondary metabolism of angiosperm including Salvia medicinal plants. The protein environment of the N-terminal class II active site stabilizes the carbocation intermediates and maintains the catalytic activity of angiosperm class II diTPS. The virtual modeling and mutagenesis of the class II diTPS of Salvia miltiorrhiza (SmCPS) were accomplished to illuminate the catalytic activity of SmCPS. Terminal truncations and point mutations established the role of the βγ domain and α domain, i.e., they facilitate the flexible conformational change of the class II active site after substrate binding. E203 and K238 in the N-terγ domain of SmCPS1are functional in the substrate binding and conformational transition and might be essential in catalysis. Similar to other CPSs, the ensuing protonation of the GGPP substrate and coordination of the diphosphate group are governed by highly conserved residues in the DxDD motif of SmCPS, e.g., D372 of CPS1. Moreover, F256 andY505stabilize the carbocation and control the enzymatic activity during CPP formation. The amino acids of the predicted active sites, despite under purifying selection, vary greatly, corresponding to the functional flexibility of angiosperm CPSs. Molecular phylogeny and evolution analyses suggest early and ongoing evolution of labdane-related diterpenoid metabolism in angiosperm.

Ky words: Copalyl diphosphate synthase, Mutagenesis, Salvia, Molecular phylogeny, Evolution.

Abbreviations: CPP, copalyl diphosphate; CPS, CPP synthase; Ta, wheat (Triticum aestivum); Hv, barley (Hordeum vulgare); Os, rice (Oryza sativa); At, Arabidopsis thaliana; KS, ent-kaurene synthase; AgAS, grand fir (Abies grandis) abietadiene synthase; GC-MS, gas chromatography with mass spectrometric detection; GGPP, ((E,E,E)-geranylgeranyl diphosphate; PBS, phosphate buffered saline.

¹Biotechnology Institute, School of Environment, Dalian Jiaotong University, Dalian 116028, China

²Department of Metabolic Biology, John Innes Centre, Norwich NR4 7UH, UK

³Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing 100193, China

^*Corresponding author’s email: hao@djtu.edu.cn