Biochemical characterization of phosphoenolpyruvate carboxykinases from multiple species of brown algae.

Phosphoenolpyruvate carboxykinase (PEPCK) is involved in the conversion of phosphoenolpyruvate (PEP) to oxaloacetate (OAA). In addition to playing a role in gluconeogenesis in various organisms, PEPCK also functions in the C4 cycle to concentrate CO2 for photosynthesis in some C4 plants. Brown algae harbor genes related to the C4 cycle, including the PEPCK gene, and are proposed to employ a C4 cycle-like pathway. However, little is known about the CO2-concentrating mechanisms and the properties of the enzymes involved in brown algae. Here, we obtained soluble recombinant PEPCKs of five brown algae and carried out biochemical analyses. The five PEPCKs were ATP-dependent and displayed similar or higher specific activities compared with their counterparts from other organisms. Phosphoenolpyruvate carboxykinase from Ishige okamurae (Io-PEPCK) exhibited the highest specific activity in both carboxylation and decarboxylation directions, with values of 48.4 and 63.3 μmol · min-1 · mg-1, respectively. Additionally, Io-PEPCK displayed a kcat/KmHCO3 value of 9.2 × 103 · M-1 · s-1, much higher than those of previously characterized PEPCKs. The response of PEPCK activity to various metabolites showed that citrate and malate inhibited the carboxylation but promoted the decarboxylation activity of Io-PEPCK. Various ATP concentrations resulted in different degrees of inhibition on the carboxylation activity of PEPCK, suggesting that ATP concentration potentially regulates PEPCK activity in brown algae. The analysis of cell extracts from I. okamurae suggested that PEPCK rather than PEPC dominates the carboxylation in this brown alga. Based on previous knowledge and the results presented here, a model for a C4 cycle-like pathway in brown algae has been proposed.