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

Abstract

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 ( Sy FumC) were 7.5 and 30 °C, respectively. The K m of Sy FumC for l- malate was higher than for fumarate. Furthermore, Sy FumC activity was strongly inhibited by citrate and succinate, consistent with fumarases in other organisms. Substitution of alanine by glutamate at position 314 of Sy FumC changed the k cat for fumarate and l- malate. In addition, the inhibitory effects of citrate and succinate on Sy FumC activity were alleviated. Phylogenetic analysis revealed cyanobacterial fumarase clades divided in non-nitrogen-fixing cyanobacteria and nitrogen-fixing cyanobacteria. Sy FumC was thus biochemically characterised, including identification of an amino acid residue important for substrate affinity and enzymatic activity.

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

“…Synechocystis also encodes a succinyl-CoA synthetase complex (SucC/SucD), which probably catalyses the reversible conversion of succinate into succinyl-CoA in cyanobacteria [54], required for biosynthesis of methionine and lysine. Several recent papers have investigated the enzymatic properties of TCA enzymes conserved between cyanobacteria and heterotrophic bacteria [55][56][57]. In contrast with many heterotrophic bacteria, Synechocystis citrate synthase (GltA) was shown only to catalyse generation of citrate, not its cleavage.…”

Fumarase From Cyanidioschyzon merolae Stably Shows High Catalytic Activity for Fumarate Hydration Under High Temperature Conditions

“…It is suggested that TeFum also becomes active at these growth temperatures. The optimum pH for CmFUM (pH 7.5 for fumarate hydration; pH 8.5 for l-malate dehydration) and TeFum (pH 7.0 for fumarate hydration; pH 7.5 for l-malate dehydration) were approximately the same as those of Class ІІ Fums from other organisms (pH 6.5-8.5, seven species: R. oryzae, Synechocystis 6803, Saccharomyces cerevisiae, S. solfataricus, C. glutamicum, A. thaliana, and Homo sapience; Puchegger et al, 1990;Keruchenko et al, 1992;Genda et al, 2006;Song et al, 2011;Zubimendi et al, 2018;Ajalla Aleixo et al, 2019;Katayama et al, 2019; Figures 2B,D). Intercellular pH of C. merolae and cyanobacteria is maintained near neutral where CmFUM and TeFum show enzymatic activities (Coleman and Colman, 1981;Zenvirth et al, 1985;Mangan et al, 2016).…”

“…Fums from A. thaliana (mitochondrial Fum;Zubimendi et al, 2018) and Synechocystis 6803 (Katayama et al, 2019), there was a significant difference between the optimum pH for fumarate hydration and l-malate dehydration in CmFUM and TeFum (particularly CmFUM; Figures 2B,D). Therefore, we can regulate the equilibrium of the reaction catalyzed by CmFUM by adjusting the pH.…”

“…Like Class ІІ Fums from other organisms (A. thaliana, Synechocystis 6803, C. glutamicum, and H. sapience; Genda et al, 2006;Zubimendi et al, 2018;Ajalla Aleixo et al, 2019;Katayama et al, 2019), CmFUM and TeFum preferentially catalyze fumarate hydration rather than l-malate dehydration ( Table 1). The K m of CmFUM (0.27 mM) and TeFum (0.14 mM) for fumarate were within the range of most Class ІІ enzymes (0.03-3.07 mM, nine species: E. coli, C.…”

“…Bootstrap value obtained by 500 replications indicates the reliability of each branch. Katayama et al, 2019; Table 1). The k cat of CmFUM (235 s −1 ) and TeFum (37 s −1 ) for fumarate were within the range of Class ІІ Fums (21-513 s −1 , four species: C. glutamicum, Synechocystis 6803, A. thaliana, and H. sapience, Lin et al, 2018;Zubimendi et al, 2018;Ajalla Aleixo et al, 2019;Katayama et al, 2019; Table 1).…”