Zhou Wenxu, Nes W David
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.
Arch Biochem Biophys. 2003 Dec 1;420(1):18-34. doi: 10.1016/j.abb.2003.08.029.
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 Delta(24(25))-bond (cycloartenol; K(m) 35 microM and k(cat) 0.001s(-1)) and Delta(24(28))-bond (24(28)-methylenelophenol; K(m) 28 microM 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 Delta(24(28))Z-ethylidene, Delta(24(28))E-ethylidene, and Delta(25(27))-24 beta-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, Delta(24(24))<Delta(24(28)) and nucleus, Delta(7)<Delta(8)<Delta(5)=9,19-cyclopropane. The 14 alpha-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')-3 beta,24 beta-dienol by SMT2, K(m) of 15 microM, 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 microM, 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. [28E-2H]- and [28Z-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 Delta(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.
拟南芥甾醇甲基转移酶2（SMT2）cDNA在大肠杆菌中表达产生一种天然蛋白，纯化至同质后，在SDS-PAGE上具有预测的约40 kDa分子量，可识别拟南芥合成的具有Δ(24(25))键（环阿屯醇；Km 35 μM，kcat 0.001 s-1）和Δ(24(28))键（24(28)-亚甲基环菠萝醇；Km 28 μM，kcat 0.01 s-1）的天然甾醇。环阿屯醇转化为单一烯烃产物——24(28)-亚甲基环阿屯醇，而24(28)-亚甲基环菠萝醇转化为具有Δ(24(28))Z-亚乙基、Δ(24(28))E-亚乙基和Δ(25(27))-24β-乙基侧链的三种立体化学相关产物的混合物。活性所必需的结构决定因素是C-3和C-24处的亲核特征。甾醇底物中的双键位置按照以下顺序影响催化效率：侧链，Δ(24(24))<Δ(24(28))；核，Δ(7)<Δ(8)<Δ(5)=9,19-环丙烷。14α-甲基对催化有害，降低了环阿屯醇作为底物的适用性。基于底物活性和产物分布，使用SMT催化反应的底物（26,27-脱氢酵母甾醇：26,27-DHZ）和中间体（25-氮杂环阿屯醇：25-AC）类似物进一步探究SMT作用。SMT2将26,27-DHZ C-甲基化生成26-高胆甾-8(9),23(24)E,26(26')-三烯醇以及26-高胆甾-8(9),26(26')-3β,24β-二烯醇，Km为15 μM，kcat为0.001 s-1。此外，26,27-DHZ作为基于机制的不可逆抑制剂，导致SMT2的特异性共价修饰，表现出Ki为49 μM，k(inact)为0.009 s-1，分配比为0.11。用酵母甾醇、24(28)-亚甲基环菠萝醇对26,27-DHZ进行底物保护，以及用Ki为20 nM的可逆抑制剂25-AC对第一次和第二次C(1)-转移活性进行类似抑制，表明这些类似物在同一活性位点相互作用。[28E-2H]-和[28Z-2H]24(28)-亚甲基羊毛甾醇与腺苷甲硫氨酸配对，酶生成的24-乙基烯烃中2H掺入的差异支持一种反机制。结果表明植物SMT2对Δ(24(25))-甾醇具有位置特异性底物特异性，并且包含一个单一活性中心，通过类似于真菌SMT1的底物反应通道催化连续的C(1)-转移活性。
