Mangla A T, Nes W D
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409, USA.
Bioorg Med Chem. 2000 May;8(5):925-36. doi: 10.1016/s0968-0896(00)00040-7.
The membrane-bound sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii, a non-photosynthetic, yeast-like alga, was found to C-methylate appropriate delta24(25)-sterol acceptor molecules to delta25(27)-24beta-methyl products stereoselectively. Incubation with pairs of substrates--[2H3-methyl]AdoMet and cycloartenol, and AdoMet and [27-(13)C]lanosterol--followed by 1H and 13C NMR analysis of the isotopically labeled products demonstrated the si-face (beta-face attack) mechanism of C-methylation and the regiospecificity of delta25(27)-double bond formation from the pro-Z methyl group (C27) on lanosterol. The enzyme has a substrate preference for a sterol with a 3beta-hydroxyl group, a planar nucleus and a side chain oriented into a 'right-handed' structure (20R-chirality) characteristic of the native substrate, cycloartenol. The apparent native molecular weight of the SMT was determined to be approximately 154,000, as measured by Superose 6 FPLC. A series of sterol analogues which contain heteroatoms substituted for C24 and C25 or related structural modifications, including steroidal alkaloids, havs been used to probe further the active site and mechanism of action of the SMT enzyme. Sterol side chains containing isoelectronic modifications of a positively charged moiety in the form of an ammonium group substituted for carbon at C25, C24, C23 or C22 are particularly potent non-competitive inhibitors (Ki for the most potent inhibitor tested, 25-azacycloartanol, was ca. 2 nM, four orders of magnitude less than the Km for cycloartenol of 28 microM), supporting the intermediacy of the 24-methyl C24(25)-carbenium ion intermediate. Ergosterol, but neither cholesterol nor sitosterol, was found to inhibit SMT activity (Ki = 80 microM). The combination of results suggests that the interrelationships of substrate functional groups within the active center of a delta24(25) to delta25(27) 24beta-methyl-SMT could be approximated thereby allowing the rational design of C-methylation inhibitors to be formulated and tested.
从威克汉姆原藻（一种非光合的、酵母样藻类）中分离出的膜结合甾醇甲基转移酶（SMT），能将合适的δ24(25)-甾醇受体分子立体选择性地C-甲基化为δ25(27)-24β-甲基产物。将底物对——[2H3-甲基]腺苷甲硫氨酸和环阿屯醇，以及腺苷甲硫氨酸和[27-(13)C]羊毛甾醇——一起孵育，然后对同位素标记产物进行1H和13C NMR分析，结果表明C-甲基化的si-面（β-面进攻）机制以及羊毛甾醇上来自前Z-甲基（C27）的δ25(27)-双键形成的区域特异性。该酶对具有3β-羟基、平面核以及侧链呈天然底物环阿屯醇所特有的“右手”结构（20R-手性）的甾醇具有底物偏好性。通过Superose 6 FPLC测定，SMT的表观天然分子量约为154,000。一系列含有取代C24和C25的杂原子或相关结构修饰的甾醇类似物，包括甾体生物碱，已被用于进一步探究SMT酶的活性位点和作用机制。含有以铵基团形式在C25、C24、C23或C22处取代碳的带正电荷部分的等电子修饰的甾醇侧链是特别有效的非竞争性抑制剂（测试的最有效抑制剂25-氮杂环阿屯醇的Ki约为2 nM，比环阿屯醇的Km值28 μM低四个数量级），这支持了24-甲基C24(25)-碳正离子中间体的中间体性质。发现麦角甾醇能抑制SMT活性（Ki = 80 μM），而胆固醇和谷甾醇则不能。这些结果综合起来表明，δ24(25)至δ25(27) 24β-甲基-SMT活性中心内底物官能团的相互关系可以由此得到近似，从而能够设计并测试C-甲基化抑制剂。
