Genkov Todor, Spreitzer Robert J
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, USA.
J Biol Chem. 2009 Oct 30;284(44):30105-12. doi: 10.1074/jbc.M109.044081. Epub 2009 Sep 4.
The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.
叶绿体酶核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）催化光合二氧化碳固定的限速步骤。随着对其结构-功能关系以及氧气竞争性抑制的深入了解，有可能通过基因工程提高农业生产力和可再生能源产量。叶绿体编码的大亚基构成活性位点，但核编码的小亚基也会影响催化效率和二氧化碳/氧气特异性。为了进一步明确小亚基在Rubisco功能中的作用，通过转化缺乏小亚基基因家族的莱茵衣藻突变体，将所有小亚基中10个最保守的残基替换为丙氨酸。所有突变株都能进行光合作用生长，这表明这些残基对功能都不是必需的。其中三个替换几乎没有影响（S16A、P19A和E92A），一个主要影响全酶稳定性（L18A），其余的影响催化作用，同时伴有一定程度的结构不稳定性（Y32A、E43A、W73A、L78A、P79A和F81A）。Y32A和E43A导致二氧化碳/氧气特异性降低。基于莱茵衣藻Rubisco的X射线晶体结构，除一个保守残基（Glu-92）外，其余保守残基均与大亚基接触，并聚集在大亚基α-螺旋8的氨基或羧基末端附近，α-螺旋8是α/β-桶状活性位点的一个结构元件。小亚基残基Glu-43和Trp-73确定了活性位点α-螺旋8与β链A和B之间高度可变的小亚基环之间可能存在的结构联系，这也会影响Rubisco的二氧化碳/氧气特异性。
