García-Murria María-Jesús, Karkehabadi Saeid, Marín-Navarro Julia, Satagopan Sriram, Andersson Inger, Spreitzer Robert J, Moreno Joaquín
Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de València, Av. Dr Moliner 50, Burjassot, València E-46100, Spain.
Biochem J. 2008 Apr 15;411(2):241-7. doi: 10.1042/BJ20071422.
Proximal Cys(172) and Cys(192) in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys(172) has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys(172) and Cys(192) by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The V(c) (V(max) for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the K(m) for CO(2) and O(2) was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 degrees C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 degrees C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of beta-strand 1 of the alpha/beta-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.
光合作用酶核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco；EC 4.1.1.39）大亚基中的近端半胱氨酸（Cys）172和Cys192在蓝细菌、藻类和高等植物中具有进化保守性。已表明Cys172的突变会影响体外Rubisco的氧化还原特性，并在胁迫条件下延迟体内该酶的降解。在此，我们报告了用丝氨酸取代Cys172和Cys192对莱茵衣藻Rubisco的催化特性、热稳定性和三维结构的影响。与野生型酶相比，C172S取代最显著的影响是特异性因子增加了11%。C192S Rubisco的特异性因子未改变。对于C172S酶，V(c)（羧化反应的V(max)）与野生型Rubisco相似，但对于C192S Rubisco，V(c)约低30%。相比之下，C192S和野生型酶对CO₂和O₂的K(m)相似，但C172S酶的K(m)明显更高（约为两倍）。C172S Rubisco的临界变性温度比野生型Rubisco低约2℃，在55℃时变性速率明显更高，而C192S Rubisco对温度变性的敏感性仅略高于野生型酶。X射线晶体结构表明，C172S突变导致α/β桶状结构β链1的主链骨架原子发生位移，影响了多个氨基酸侧链。这可能导致C172S具有特殊的催化特性。相比之下，C192S突变不会产生类似的结构扰动。
