Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis

Biochimica Et Biophysica Acta
Maristela PereiraW David Nes

Abstract

The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502Da. The enzymatic C24-methylation gave a K(mapp) of 38microM and k(catapp) of 0.14min(-1). Quite unexpectedly, "plant" cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-(13)C]- or [24-(2)H]cycloartenol with PbSMT and analysis of the enz...Continue Reading

Citations

Feb 4, 2014·Evidence-based Complementary and Alternative Medicine : ECAM·Hsueh-Ling ChengChang-Hung Chou
Sep 29, 2011·The Journal of Steroid Biochemistry and Molecular Biology·Katja Kristan, Tea Lanišnik Rižner
Feb 21, 2013·Microbes and Infection·Daciene de Arruda GrossklausCélia Maria de Almeida Soares
Jan 7, 2016·PLoS Neglected Tropical Diseases·Felipe Souto AraújoMaristela Pereira
Apr 9, 2017·Applied Microbiology and Biotechnology·Ashraf S A El-SayedGul Shad Ali
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Jan 29, 2020·Future Microbiology·Luciane S da SilvaRoosevelt A da Silva
Nov 3, 2011·PLoS Genetics·Christopher A DesjardinsChristina A Cuomo
Sep 10, 2011·Chemical Reviews·W David Nes
Oct 8, 2021·Planta·Yiyang ChenXiaowei Du
Oct 16, 2010·Archives of Biochemistry and Biophysics·Kulothungan GanapathyW David Nes

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