Sterol methyltransferase2: purification, properties, and inhibition

Abstract

Expression of the Arabidopsis sterol methyltransferase2 (SMT2) cDNA in Escherichia coli yields a native protein, when purified to homogeneity, has the predicted molecular mass ca. 40 kDa on SDS–PAGE and recognizes native sterols synthesized by Arabidopsis with a Δ 24(25)–bond (cycloartenol; K m 35 μM and k cat 0.001 s −1) and Δ 24(28)-bond (24(28)-methylenelophenol; K m 28μM and k cat 0.01 s −1). Cycloartenol was converted to a single olefinic product-24(28)-methylenecycloartanol whereas 24(28)-methylenelophenol was converted to a mixture of three stereochemically related products with the Δ 24(28) Z-ethylidene, Δ 24(28) E-ethylidene, and Δ 25(27)-24β-ethyl side chains. Structural determinants essential to activity were the nucleophilic features at C-3 and C-24. The double bond position in the sterol substrate influenced catalytic efficiency according to the order: side chain, Δ 24(24)<Δ 24(28) and nucleus, Δ 7<Δ 8<Δ 5=9,19-cyclopropane. The 14α-methyl group was harmful to catalysis, reducing the suitability of cycloartenol as a substrate. On the basis of substrate activity and product distribution, SMT action was probed further using substrate (26,27-dehydrozymosterol: 26,27-DHZ) and intermediate (25-azacycloartenol: 25-AC) analogs of the SMT-catalyzed reactions. 26,27-DHZ was C-methylated to 26-homocholesta-8(9), 23(24) E, 26(26 ′)-trienol as well as 26-homocholesta-8(9),26(26 ′)-3β,24β-dienol by SMT2, K m of 15 μM, k cat of 0.001 s −1. In addition, 26,27-DHZ acted as a mechanism-based irreversible inhibitor that results in the specific covalent modification of SMT2, exhibiting K i of 49 μM, k inact of 0.009 s −1 and partition ratio of 0.11. Substrate protection with zymosterol, 24(28)-methylenelophenol against 26,27-DHZ and similar inhibition of the first and second C 1-transfer activities by the reversible inhibitor 25-AC of K i 20 nM suggested the analogs interacted at the same active site. [28 E- 2H]- and [28 Z- 2H]24(28)-methylenelanosterols were paired with AdoMet and differences of 2H-incorporation in the enzyme-generated 24-ethyl olefins supported an antimechanism. The results suggest plant SMT2 has a position-specific substrate specificity for Δ 24(25)-sterols and contains a single active center to catalyze the consecutive C 1-transfer activities by substrate reaction channels similar to the fungal SMT1.