Koo L S, Tschirret-Guth R A, Straub W E, Moënne-Loccoz P, Loehr T M, Ortiz de Montellano P R
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
J Biol Chem. 2000 May 12;275(19):14112-23. doi: 10.1074/jbc.275.19.14112.
CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 degrees C. UV-visible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin state at higher temperatures. Styrene only causes a small spin state shift, but T(1) NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H(2)O(2)-dependent epoxidation of styrene, cis-beta-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 degrees C for 90 min. Site-specific mutagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state. Topological analysis by reaction with aryldiazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away. CYP119 is very slowly reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants. Protein melting curves indicate that the thermal stability of CYP119 does not depend on the iron spin state or the active site architecture defined by the threonine residues. Independence of thermal stability from active site structural factors should facilitate the engineering of novel thermostable catalysts.
来自嗜热栖热菌的CYP119是首个嗜热细胞色素P450，在高达85摄氏度时仍保持稳定。紫外可见光谱和共振拉曼光谱表明，该酶处于低自旋状态，在较高温度下仅适度转变为高自旋状态。苯乙烯仅引起较小的自旋状态变化，但T(1)核磁共振研究证实苯乙烯结合在活性位点。CYP119催化苯乙烯、顺式-β-甲基苯乙烯和顺式二苯乙烯的H(2)O(2)依赖性环氧化反应，并保留立体化学结构。这种催化活性在80摄氏度下预孵育90分钟后仍保持稳定。定点诱变表明，苏氨酸-213具有重要催化作用，苏氨酸-214有助于控制铁的自旋状态。通过与芳基重氮化合物反应进行的拓扑分析表明，苏氨酸-213位于吡咯环A和B上方，靠近铁原子，而苏氨酸-214则相距一定距离。CYP119被恶臭假单胞菌铁氧还蛋白和恶臭假单胞菌铁氧还蛋白还原酶还原的速度非常缓慢，但这些蛋白质支持苏氨酸-214突变体的催化周转。蛋白质熔解曲线表明，CYP119的热稳定性不依赖于铁的自旋状态或由苏氨酸残基定义的活性位点结构。热稳定性与活性位点结构因素的独立性应有助于新型热稳定催化剂的工程设计。
