Plant cyclopropylsterol-cycloisomerase: key amino acids affecting activity and substrate specificity

Abstract

The enzyme CPI (cyclopropylsterol-cycloisomerase) from the plant sterol pathway catalyses the cleavage of the 9β,19-cyclopropane ring of the 4α-methyl-cyclopropylsterol cycloeucalenol to produce the Δ8-sterol obtusifoliol. Randomly mutated plasmids carrying the Arabidopsis thaliana cpi gene were screened for inactive CPI mutant enzymes on the basis of their ability to genetically complement a Saccharomyces cerevisiae erg7 (defective in oxidosqualene cyclase) ergosterol auxotroph grown in the presence of exogenous cycloeucalenol, and led to the identification of four catalytically important residues. Site-directed mutagenesis experiments confirmed the role of the identified residues, and demonstrated the importance of selected acidic residues and a conserved G108NYFWTHYFF117 motif. The mutated isomerases were assayed both in vivo by quantification of cycloeucalenol conversion into ergosterol in erg7 cells, and in vitro by examination of activities of recombinant AtCPI (A. thaliana CPI) mutants. These studies show that Gly28, Glu29, Gly108 and Asp260 are crucial for CPI activity and that an hydroxy function at residue 113 is needed for maximal substrate affinity and CPI activity. CPI is inactive on upstream 4α,β-dimethyl-cyclopropylsterol precursors of phytosterols. The single mutation W112L generates a CPI with an extended substrate specificity, that is able to convert 4α,β-dimethyl-cyclopropylsterols into the corresponding Δ8 products. These findings provide insights into the molecular basis of CPI activity and substrate specificity.

Molecular Docking and Molecular Dynamics Studies on Selective Synthesis of α-Amyrin and β-Amyrin by Oxidosqualene Cyclases from Ilex Asprella

“…α-Amyrin was the main product of IaAS1, and the ratio of α-amyrin to β-amyrin was about 4:1. β-Amyrin was the main product of IaAS2, and the ratio of α-amyrin to β-amyrin was about 1:19 [16]. Several site-directed mutations of other OSCs (AsAS: β-amyrin synthase of Avena strigose [18], AtCYC: Cycloartenol synthase of Arabidopsis thaliana [19], AtCBS: Cucurbitadienol synthase of Arabidopsis thaliana [20], AtCPI: Cyclopropylsterol-cycloisomerase of Arabidopsis thaliana [21], AtLSSl: Lanosterol synthase of Arabidopsis thaliana [22], AtLUP1: Lupeol synthase 1 of Arabidopsis thaliana [18], CcLSS: Lanosterol cyclase of Cephalosporium caerulens [23], EtAS: β-amyrin synthase of Euphorbia tirucalli [24,25,26,27], OeLS: Lupeol synthase of Olea europaea [28], PgAS: β-amyrin Synthase of Panax ginseng [28], and ScLSS: Lanosterol cyclase of Saccharomyces cerevisiae [29,30,31,32,33,34,35,36,37]) have been performed (Supplementary Table S1). Multi-sequence alignment of sequences of IaAS1, IaAS2, human lanosterol synthase and seven enzymes from plants (AsAS, AtCYC, AtLSS1, AtLUP1, EtAS, O2LS, PgAS) provides key information for identifying the interface of enzyme-substrate/intermediate/product interaction (Supplementary Figure S1).…”