Department of Microbiology, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand; Graduate School of Environmental and Human Sciences, Meijo University, Nagoya, Aichi, 468-8502, Japan.
Department of Microbiology, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand.
Arch Biochem Biophys. 2019 Sep 15;672:108059. doi: 10.1016/j.abb.2019.07.024. Epub 2019 Jul 26.
Fructose-1,6-bisphosphate aldolase (FBA) is a key metabolic enzyme, which is involved in glycolysis, gluconeogenesis and the Calvin cycle. The distinct physiological roles of FBAs in various organisms have been reported; however, in cyanobacteria, the functional characterization of FBAs and investigation of the intracellular dynamics of FBAs largely remains unknown. Here, we utilized a two-step chromatographic technique to identify a class I FBA (CI-FBA), which we named H2846. H2846 was induced by salt stress in the halotolerant cyanobacterium Halothece sp. PCC 7418 (hereafter referred to as Halothece 7418). Phylogenetic analysis showed that H2846-like CI-FBAs existed mainly in cyanobacterial species that inhabit hypersaline environments. Subcellular fractionation revealed that H2846 localized in the cytosolic and periplasmic spaces and size-exclusion chromatography suggested that H2846 formed a homohexamer. The CI-FBA activity of recombinant H2846-mediated cleavage of fructose bisphosphate (FBP) was characterized using a coupled enzymatic assay. This analysis allowed us to determine the K and V values of recombinant H2846, which were then compared to previously reported K and V values of several FBAs. Our data suggested that H2846 was likely responsible for the salt stress-induced CI-FBA activity from the total soluble protein extracts derived from Halothece 7418 cells. Moreover, heterologous expression of H2846 but not H2847, a class II FBA (CII-FBA), conferred salt stress tolerance to the salt-sensitive freshwater cyanobacterium, Synechococcus elongatus PCC 7942, which only contains the CII-FBA, S1443. S. elongatus PCC 7942 with a S1443 gene deletion was complemented by H2847 expression, but was not complemented by expression of H2846. Taken together, these results indicate the functional differences between two distinct sets of FBAs in cyanobacteria. H2846 is an active CI-FBA that contributes to the mechanism of salt stress tolerance in Halothece 7418.
果糖-1,6-二磷酸醛缩酶（FBA）是一种关键的代谢酶，参与糖酵解、糖异生和卡尔文循环。不同生物体中 FBA 的独特生理作用已有报道；然而，在蓝藻中，FBA 的功能特征和细胞内 FBA 动力学的研究在很大程度上仍然未知。在这里，我们利用两步色谱技术鉴定了一种 I 类 FBA（CI-FBA），我们将其命名为 H2846。H2846 是在耐盐蓝藻盐单胞菌 PCC 7418（简称盐单胞菌 7418）中由盐胁迫诱导产生的。系统发育分析表明，H2846 样 CI-FBAs 主要存在于栖息在高盐环境中的蓝藻物种中。亚细胞分级分离表明 H2846 定位于细胞质和周质空间，尺寸排阻色谱表明 H2846 形成同源六聚体。使用偶联酶测定法对重组 H2846 介导的果糖二磷酸（FBP）裂解的 CI-FBA 活性进行了表征。该分析使我们能够确定重组 H2846 的 K 和 V 值，然后将其与以前报道的几种 FBA 的 K 和 V 值进行比较。我们的数据表明，H2846 可能负责从盐单胞菌 7418 细胞的总可溶性蛋白提取物中诱导盐胁迫下的 CI-FBA 活性。此外，H2846 的异源表达而非 II 类 FBA（CII-FBA）的 H2847 的表达，赋予了对仅含有 CII-FBA 的盐敏感淡水蓝藻集胞藻 PCC 7942 的盐胁迫耐受性。集胞藻 PCC 7942 中 S1443 基因缺失的互补由 H2847 的表达来完成，但由 H2846 的表达来完成。总之，这些结果表明了蓝藻中两种不同 FBA 之间的功能差异。H2846 是一种活跃的 CI-FBA，有助于盐单胞菌 7418 耐盐机制。
