Rational engineering of Thermus thermophilus cytochrome c552 to a thermally tolerant artificial peroxidase
Rational engineering of Thermus thermophilus cytochrome c552 to a thermally tolerant artificial peroxidase
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DOI：
10.1039/b924365h
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发表时间：
2010-01-01
影响因子：
4
通讯作者：
Watanabe, Yoshihito
中科院分区：
文献类型：
--
作者：
Nakajima, Hiroshi;Ramanathan, Kalaivani;Watanabe, Yoshihito
在以前的研究中，我们构建了一个原型耐热人工过氧化物酶的电子转移蛋白，细胞色素c（552）从嗜热栖热菌，并证明，工程蛋白质从嗜热菌可能是一个有前途的方法来生产人工酶的实际用途。在本研究中，进一步改善的原型（V49 D/M69 A突变体）的酶活性和耐热性已实现了连续的修改的基础上形成的活性中间体在过氧化物酶反应的详细分析。光谱研究表明，V49 D/M69 A的主要活性中间体是氧代铁基血红素，其蛋白质基团主要位于Tyr 45上。EPR研究中的磁功率饱和测量表明，氧代铁基血红素和酪氨酰自由基之间的磁耦合很小。这一结果表明，氧代铁血红素和酪氨酰基自由基作为孤立的氧化剂。动力学研究表明，在活性中间体中分离的氧代铁基血红素组分可以通过与H2 O2反应而成为血红素降解的前体。在V49 D/M69 A上用苯丙氨酸取代Tyr 45导致自由基在蛋白质上的离域化，并且增加了中间体中的氧代铁基血红素和蛋白质自由基之间的磁耦合。通过用色氨酸取代Tyr 45实现了氧代铁基血红素和自由基之间的更强的磁耦合，这类似于在一些过氧化氢酶-过氧化物酶的活性中间体中发现的一个色氨酸基。Tyr 45突变体的蛋白质自由基中间体表现出相对较高的反应性，以有机底物比H2 O2相比，基础突变体，V49 D/M69 A，这是优选的，以抑制血红素降解过程。这反映在过氧化物酶反应中观察到Tyr 45突变体的酶活性和耐热性改善。
In a previous study, we constructed a prototype thermally tolerant artificial peroxidase from an electron transfer protein, cytochrome c(552) from Thermus thermophilus, and demonstrated that engineering of proteins from thermophiles could be a promising methodology to produce artificial enzymes for practical use. In the present study, further improvement of the prototype (the V49D/M69A mutant) in enzymatic activity and thermal tolerance has been achieved by successive modifications based on detailed analyses of an active intermediate formed in the peroxidase reaction. Spectroscopic studies revealed that the major active intermediate of V49D/M69A was an oxo-ferryl heme with a protein radical predominantly localized on Tyr45. The magnetic power saturation measurement in EPR studies showed little magnetic coupling between the oxo-ferryl heme and the tyrosyl radical. This result indicated that the oxo-ferryl heme and the tyrosyl radical served as isolated oxidants. Kinetics studies indicated that the isolated oxo-ferryl heme component in the active intermediate could be a precursor to heme degradation by the reaction with H2O2. Replacement of Tyr45 with phenylalanine on V49D/M69A resulted in delocalization of the radical over the protein and increased magnetic coupling between the oxo-ferryl heme and the protein radical in the intermediate. The stronger magnetic coupling between the oxo-ferryl heme and the radical was achieved by replacement of Tyr45 with tryptophan, which was similar to a tryptophanyl radical found in active intermediates of some catalase-peroxidases. The protein radical intermediates of the Tyr45 mutants exhibited relatively higher reactivity to an organic substrate than H2O2 in comparison to the basal mutant, V49D/M69A, which was preferable to suppress the heme degradation process. This was reflected in the improved enzymatic activity and thermal tolerance observed for the Tyr45 mutants in the peroxidase reaction.