Wüst M, Little D B, Schalk M, Croteau R
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340, USA.
Arch Biochem Biophys. 2001 Mar 1;387(1):125-36. doi: 10.1006/abbi.2000.2248.
Limonene enantiomers and substrate analogs, including specifically fluorinated derivatives, were utilized to probe active site interactions with recombinant (-)-(4S)-limonene-3-hydroxylase (CYP71D13) and (-)-(4S)-limonene-6-hydroxylase (CYP71D18) from mint (Mentha) species. (-)-(4S)-Limonene is hydroxylated by both enzymes at the designated C3- and C6-allylic positions, with strict regio- and stereospecificity and without detectable allylic rearrangement, to give the corresponding products (-)-trans-isopiperitenol and (-)-trans-carveol. CYP71D13-catalyzed hydroxylation of (+)-(4R)-limonene also yields the corresponding trans-3-hydroxylated product ((+)-transisopiperitenol); however, the C6-hydroxylase converts (+)-(4R)-limonene to a completely different product profile dominated by the enantiopure cis-6-hydroxylated product (+)-cis-carveol along with several minor products, including both enantiomers of the trans-6-hydroxylated product ((+/-)-trans-carveol), indicating allylic rearrangement during catalysis. These results demonstrate that the regiospecificity and facial stereochemistry of oxygen insertion is dictated by the absolute configuration of the substrate. Fluorinated limonene analogs are also tightly bound by both enzymes and hydroxylated at the topologically congruent positions in spite of the polarizing effect of the fluorine atom on substrate reactivity. This strict retention of oxygenation geometry suggests a rigid substrate orientation imposed by multiple hydrophobic active site contacts. Structurally simplified substrate analogs are hydroxylated at slower rates and with substantial loss of regiospecificity, consistent with a loss of active site complementarity. Evaluation of the product profiles generated allowed assessment of the role of hydrophobic contacts in orienting the substrate relative to the activated oxygen species.
柠檬烯对映体和底物类似物，包括特定的氟化衍生物，被用于探究与薄荷（Mentha）属植物的重组（-）-（4S）-柠檬烯-3-羟化酶（CYP71D13）和（-）-（4S）-柠檬烯-6-羟化酶（CYP71D18）的活性位点相互作用。（-）-（4S）-柠檬烯在指定的C3-和C6-烯丙基位置被这两种酶羟基化，具有严格的区域和立体特异性，且没有可检测到的烯丙基重排，生成相应的产物（-）-反式异胡薄荷醇和（-）-反式香芹醇。CYP71D13催化的（+）-（4R）-柠檬烯羟基化也产生相应的反式-3-羟基化产物（（+）-反式异胡薄荷醇）；然而，C6-羟化酶将（+）-（4R）-柠檬烯转化为完全不同的产物谱，主要是对映体纯的顺式-6-羟基化产物（+）-顺式香芹醇以及几种次要产物，包括反式-6-羟基化产物（（+/-）-反式香芹醇）的两种对映体，这表明催化过程中发生了烯丙基重排。这些结果表明，氧插入的区域特异性和面立体化学由底物的绝对构型决定。尽管氟原子对底物反应性有极化作用，但氟化柠檬烯类似物也被这两种酶紧密结合，并在拓扑学上一致的位置被羟基化。这种严格的氧化几何构型保留表明多个疏水活性位点接触施加了刚性的底物取向。结构简化的底物类似物羟基化速率较慢，区域特异性显著丧失，这与活性位点互补性的丧失一致。对生成的产物谱进行评估可以评估疏水接触在使底物相对于活化氧物种定向方面的作用。
