Department of Microbiology , The Ohio State University , Columbus , Ohio 43210 , United States.
UCLA-DOE Institute , University of California, Los Angeles , Los Angeles , California 90095 , United States.
Biochemistry. 2019 Sep 17;58(37):3880-3892. doi: 10.1021/acs.biochem.9b00617. Epub 2019 Sep 3.
The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO via the Calvin-Benson-Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from , a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate (CABP), greatly facilitating the structure-function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure-function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.
核酮糖-1,5-二磷酸羧化酶/加氧酶（RuBisCO）及其在卡尔文-本森-巴斯汉姆（CBB）循环中通过捕获大气 CO2 的核心作用已经得到了充分的研究。此前，一种来自 的兼性厌氧细菌的 II 型 RuBisCO 已被证明可以组装成六聚体全酶。与之前关于 II 型 RuBisCO 的研究不同，该酶可以在过渡态类似物 2-羧基-D-阿拉伯糖醇 1,5-二磷酸（CABP）的存在下结晶，极大地促进了这里报道的结构-功能研究。对具有 II 型特异性残基（Ile165 和 Met331）和酶活性位点附近其他保守和半保守残基取代的突变酶的结构分析确定了细微的结构相互作用，这些相互作用可能解释了不同的 RuBisCO 酶之间的功能差异。此外，使用远缘需氧细菌宿主，进一步选择克服负酶功能的抑制突变酶也得以完成。对负突变酶和抑制突变酶 RuBisCO 的结构-功能分析强调了涉及酶四级结构不同部分的相互作用的重要性，这些相互作用影响构成 RuBisCO 羧化机制的部分反应。特别是，在亚基间界面的结构扰动似乎会影响 CO2 的添加，但不会影响酶机制的前一个步骤，即底物核酮糖-1,5-二磷酸（RuBP）的烯醇化。这一点通过负突变酶的一部分子集能够支持先前描述的硫挽救功能得到了进一步证实，该功能似乎仅依赖于酶催化类似于 RuBP 的底物烯醇化的能力。
