Morisse Samuel, Michelet Laure, Bedhomme Mariette, Marchand Christophe H, Calvaresi Matteo, Trost Paolo, Fermani Simona, Zaffagnini Mirko, Lemaire Stéphane D
From CNRS, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France, the Sorbonne Universités, Université Pierre et Marie Curie (UPMC) Universit́ Paris 06, UMR8226, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, 75005 Paris, France.
the Department of Chemistry "G. Ciamician," University of Bologna, 40126 Bologna, Italy.
J Biol Chem. 2014 Oct 24;289(43):30012-24. doi: 10.1074/jbc.M114.597997. Epub 2014 Sep 8.
In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (-335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys(227) and Cys(361). Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view.
在光合生物中，硫氧还蛋白依赖性氧化还原调节是一种成熟的机制，参与控制大量细胞过程，包括卡尔文-本森循环。实际上，该循环的11种酶中有4种在光照下通过叶绿体硫氧还蛋白控制的二硫醇/二硫化物交换而被激活。最近，几种基于蛋白质组学的方法表明，卡尔文-本森循环的酶不仅有4种，而是所有酶都可能受到氧化还原调节。在这里，我们表征了真核绿藻莱茵衣藻中卡尔文-本森循环酶磷酸甘油酸激酶（PGK1）的氧化还原特征，并且我们表明莱茵衣藻PGK1（CrPGK1）的活性受到与低中点氧化还原电位（pH 7.9时为-335 mV）形成单个调节性二硫键的抑制。发现CrPGK1氧化会影响周转数而不改变对底物的亲和力，而酶的激活似乎由f型硫氧还蛋白特异性控制。通过定点诱变、硫醇滴定、质谱分析和三维建模相结合的方法，表明调节性二硫键涉及并非严格保守的Cys(227)和Cys(361)。基于分子力学计算，提出二硫键的形成会在酶的C末端结构域施加结构限制，这可能会降低其催化效率。因此得出结论，CrPGK1可能构成另一种光调节的卡尔文-本森循环酶，在黑暗中活性较低，在光照下依赖TRX激活。还从进化的角度讨论了这些结果。
