Murataliev Marat B, Trinh Long N, Moser Lani V, Bates Robert B, Feyereisen René, Walker F Ann
Department of Chemistry, University of Arizona, Tucson, Arizona 85721-0041, USA.
Biochemistry. 2004 Feb 24;43(7):1771-80. doi: 10.1021/bi035674b.
A protein fragment of P450BM3 (residues 73-84) which participates in palmitoleate binding was subjected to scanning chimeragenesis. Amino acids 73-84, 73-78, 75-80, and 78-82 were replaced with the homologous fragments of the insect terpenoid hydroxylase CYP4C7. The four chimeric proteins, C(73-84), C(73-78), C(75-80), and C(78-82), were expressed, purified, and characterized. All the chimeric proteins contained all the cofactors and catalyzed monooxygenation of palmitate and of the sesquiterpene farnesol. Chimeragenesis altered substrate binding as shown by the changes in the amplitude of the palmitate-induced type I spectral shift. C(78-82) had monooxygenase activities close to those of P450BM3, while the rest of the chimeric proteins had monooxygenase activities that were inhibited relative to that of wild-type P450BM3. The extent of inhibition of the chimeric proteins varied depending on the substrate, and in the case of C(73-84), farnesol and palmitate oxidation was inhibited by 1 and 4 orders of magnitude, respectively. (1)H NMR spectroscopy and GC-MS were used to identify products of farnesol and palmitate oxidation. Wild-type P450BM3 and all chimeric proteins catalyzed oxidation of farnesol with formation of 9-hydroxyfarnesol and farnesol 10,11- and 2,3-epoxides. Three of the four chimeric proteins also formed a new compound, 5-hydroxyfarnesol, which was the major product in the case of C(73-78). In addition to hydroxylation of the C13-C15 atoms, the chimeric enzymes catalyze significant hydroxylation of the C10-C12 atoms of palmitate. In the case of C(78-82), the rates of formation of 11- and 12-hydroxypalmitates increased 7-fold compared to that of wild-type P450BM3 to 106 and 212 min(-)(1), respectively, while the rate of 10-hydroxypalmitate synthesis increased from zero to 106 min(-)(1). Thus, chimeragenesis of the region of residues 73-84 of the substrate binding site shifted the regiospecificity of substrate oxidation toward the center of the farnesol and palmitate molecules.
对参与棕榈油酸酯结合的P450BM3蛋白片段（第73 - 84位氨基酸残基）进行扫描嵌合诱变。将第73 - 84、73 - 78、75 - 80和78 - 82位氨基酸残基替换为昆虫类萜羟化酶CYP4C7的同源片段。表达、纯化并表征了四种嵌合蛋白C(73 - 84)、C(73 - 78)、C(75 - 80)和C(78 - 82)。所有嵌合蛋白都含有所有辅因子，并催化棕榈酸和倍半萜金合欢醇的单加氧反应。如棕榈酸诱导的I型光谱位移幅度变化所示，嵌合诱变改变了底物结合。C(78 - 82)的单加氧酶活性与P450BM3接近，而其余嵌合蛋白的单加氧酶活性相对于野生型P450BM3受到抑制。嵌合蛋白的抑制程度因底物而异，对于C(73 - 84)，金合欢醇和棕榈酸氧化分别被抑制了1个和4个数量级。使用(1)H NMR光谱和GC - MS鉴定金合欢醇和棕榈酸氧化产物。野生型P450BM3和所有嵌合蛋白催化金合欢醇氧化生成9 - 羟基金合欢醇以及金合欢醇10,11 - 环氧化物和2,3 - 环氧化物。四种嵌合蛋白中的三种还形成了一种新化合物5 - 羟基金合欢醇，在C(73 - 78)的情况下它是主要产物。除了对棕榈酸C13 - C15原子进行羟基化外，嵌合酶还催化了棕榈酸C10 - C12原子的显著羟基化。对于C(78 - 82)，11 - 羟基棕榈酸和12 - 羟基棕榈酸的生成速率分别比野生型P450BM3提高了7倍，达到106和212 min(-1)，而10 - 羟基棕榈酸的合成速率从零增加到106 min(-1)。因此，底物结合位点第73 - 84位氨基酸残基区域的嵌合诱变使底物氧化的区域特异性向金合欢醇和棕榈酸分子中心转移。
