School of Agriculture, Meiji University, 1-1-1, Higashimita, Tama-ku, Kawasaki, Kanagawa, 214-8571, Japan.
Sci Rep. 2019 Jul 23;9(1):10629. doi: 10.1038/s41598-019-47025-7.
The tricarboxylic acid cycle produces NADH for oxidative phosphorylation and fumarase [EC 4.2.1.2] is a critical enzyme in this cycle, catalysing the reversible conversion of fumarate and L-malate. Fumarase is applied to industrial L-malate production as a biocatalyst. L-malate is used in a wide range of industries such as food and beverage, pharmacy chemistry. Although the biochemical properties of fumarases have been studied in many organisms, they have not been investigated in cyanobacteria. In this study, the optimum pH and temperature of Synechocystis 6803 fumarase C (SyFumC) were 7.5 and 30 °C, respectively. The K of SyFumC for L-malate was higher than for fumarate. Furthermore, SyFumC activity was strongly inhibited by citrate and succinate, consistent with fumarases in other organisms. Substitution of alanine by glutamate at position 314 of SyFumC changed the k for fumarate and L-malate. In addition, the inhibitory effects of citrate and succinate on SyFumC activity were alleviated. Phylogenetic analysis revealed cyanobacterial fumarase clades divided in non-nitrogen-fixing cyanobacteria and nitrogen-fixing cyanobacteria. SyFumC was thus biochemically characterised, including identification of an amino acid residue important for substrate affinity and enzymatic activity.
三羧酸循环产生 NADH 用于氧化磷酸化，延胡索酸酶[EC 4.2.1.2]是该循环中的关键酶，催化富马酸和 L-苹果酸的可逆转化。延胡索酸酶作为生物催化剂应用于工业 L-苹果酸的生产。L-苹果酸广泛应用于食品饮料、制药化学等多个行业。尽管富马酸酶的生化特性已在许多生物体中进行了研究，但在蓝藻中尚未进行研究。在这项研究中，Synechocystis 6803 延胡索酸酶 C（SyFumC）的最适 pH 和温度分别为 7.5 和 30°C。SyFumC 对 L-苹果酸的 K 值高于富马酸。此外，SyFumC 活性受到柠檬酸和琥珀酸的强烈抑制，与其他生物体中的富马酸酶一致。SyFumC 中的丙氨酸被谷氨酸取代 314 位，改变了富马酸和 L-苹果酸的 k 值。此外，柠檬酸和琥珀酸对 SyFumC 活性的抑制作用得到缓解。系统发育分析显示，蓝藻富马酸酶簇分为固氮蓝藻和非固氮蓝藻。因此，对 SyFumC 进行了生化特征分析，包括确定了对底物亲和力和酶活性很重要的氨基酸残基。
