1. Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis.
- Author
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Pereira M, Song Z, Santos-Silva LK, Richards MH, Nguyen TT, Liu J, de Almeida Soares CM, da Silva Cruz AH, Ganapathy K, and Nes WD
- Subjects
- Alkylation, Amino Acid Sequence, Biocatalysis, Cholestadienols chemistry, Chromatography, Gas, Chromatography, High Pressure Liquid, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Enzyme Activation, Lanosterol chemistry, Lanosterol isolation & purification, Metabolic Networks and Pathways, Methyltransferases antagonists & inhibitors, Methyltransferases chemistry, Molecular Sequence Data, Mutagenesis, Site-Directed, Phylogeny, Phytosterols chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Reproducibility of Results, Sequence Alignment, Sequence Analysis, DNA, Substrate Specificity, Tyrosine genetics, Methyltransferases genetics, Methyltransferases metabolism, Paracoccidioides enzymology, Phytosterols biosynthesis
- 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 enzyme-generated product by a combination of (1)H and (13)CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Delta(24(25))-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K(i) 14nM) and 26,27-dehydrolanosterol (K(i) 54muM and k(inact) of 0.24min(-1)) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC(50), 30nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C(1)-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
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