点突变小麦Rca-α蛋白能够增加小麦产量潜力
核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）是植物光合作用的关键酶，然而由于Rubisco的合成和催化过程十分复杂，人们很难通过直接改造Rubisco提高植物固定CO2的能力。Rubisco活化酶（Rca）是一种可以调节Rubisco活性的酶，同时具有ATP水解酶活性，能够利用ATP将Rubisco活性位点上的抑制剂移除，是光合成途径中处于中心位置的羧化酶。RCA在植物光能利用和产量建成方面具有重要作用，已经成为作物高产育种的潜在靶点。
Rca α和β异构体存在于大多数植物物种中，α异构体与β异构体是同工酶，Rca-α仅在C端多了25-45个氨基酸，被称作CTE（C-terminal extension）区。Rca能够被ADP抑制，而Rca复合体对ADP的敏感程度受到CTE区的调控，也在硫氧还蛋白的介导下能被基质中氧化还原状态的变化所调控。
最近，巴斯夫公司的研究人员在小麦Rca-α蛋白TaRca2-α的CTE区引入了一个点突变，K428R的突变显著改变了ADP对TaRca2-α的抑制性，且不依赖于硫氧还蛋白的调控。ADP对K428R突变蛋白抑制力的降低不是ADP亲和力下降导致的，而是增加了ATP底物的亲和性以及ATP依赖的催化速度。
小麦Rca-α蛋白K428R的突变具有增加小麦光合速率，提高小麦产量的潜力。
J Biol Chem., 2019 Sep17.
A single point mutation in the C-terminal extension of wheat Rubisco activase dramatically reduces ADP inhibition via enhanced ATP binding affinity.
Author
Scafaro AP*, De Vleesschauwer D, Bautsoens N, Hannah MA, den Boer B, Gallé A, Van Rie J.
*: BASF Belgium Coordination Center-Innovation Center Gent, Belgium.
Abstract
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca) is a AAA enzyme that uses ATP to remove inhibitors from the Rubisco active site, the central carboxylation enzyme of photosynthesis. Rca α and β isoforms exist in most higher plant species, with the α isoform being identical to the β form, but having an additional 25-45 amino acids at the Rca C terminus, known as the C-terminal extension (CTE). Rca is inhibited by ADP, and the extent of ADP sensitivity of the Rca complex can be modulated by the CTE of the α isoform, particularly in relation to a disulfide bond structure that is specifically reduced by the redox-regulatory enzyme thioredoxin-f. Here, we introduced single point mutations of Lys-428 in the CTE of Rca-α from wheat (Triticum aestivum) (TaRca2-α). Substitution of Lys-428 K with Arg dramatically altered ADP inhibition, independently of thioredoxin-f regulation. We determined that the reduction in ADP inhibition in the K428R variant is not due to a change in ADP affinity, as the apparent constant for ADP binding was not altered by the K428R substitution. Rather, we observed that the K428R substitution strongly increased ATP substrate affinity and ATP-dependent catalytic velocity. These results suggest that the Lys-428 residue is involved in interacting with the γ phosphate of ATP. Considering that nucleotide-dependent Rca activity regulates Rubisco and thus photosynthesis during fluctuating irradiance, the K428R substitution could potentially provide a mechanism for boosting the performance of wheat grown in the dynamic light environments of the field.