Biochemical characterisation of fumarase C from a unicellular cyanobacterium demonstrating its substrate affinity, altered by an amino acid substitution.

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.