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 (SyFumC) were 7.5 and 30 °C, respectively. The Km 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 kcat 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.
Highlights
Fumarase, or fumarate hydratase [EC 4.2.1.2], is an enzyme in the tricarboxylic acid (TCA) cycle and is conserved in all organisms
Fluxes in the TCA cycle remain low under all conditions tested[30,33], indicating that the TCA cycle of unicellular cyanobacteria is unique among bacteria
Genome sequence analysis indicated that Synechocystis 6803 does not possess a Class I fumarase but does express a Class II fumarase, Synechocystis fumarase C (SyFumC)
Summary
Fumarate hydratase [EC 4.2.1.2], is an enzyme in the tricarboxylic acid (TCA) cycle and is conserved in all organisms. 2-oxoglutarate is converted to glutamate, followed by conversion to GABA35 which is converted to succinyl-semialdehyde, and to succinate[35] In addition to these bypasses, succinate is produced in the reductive TCA cycle in Synechocystis 6803 under dark, anaerobic conditions[36]. Substitution of the glutamate residue with lysine at position 954 restores these inhibitory effects, similar to that seen for PEPC in a nitrogen-fixing cyanobacterium[37] These recent studies demonstrate that TCA cycle enzymes in cyanobacteria possess unique properties, making biochemical characterisation of these enzymes intriguing, and indispensable for understanding their metabolism. Biochemical analysis of fumarase C from Synechocystis 6803 (SyFumC) revealed that the reversible reactions are regulated by TCA cycle metabolites and further showed the importance of the alanine residue at position 314 for substrate affinity
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