Recombinant expression, purification and osmotic regulation of in vitro activity of two enzymes constituting the Mozambique tilapia myo‐inositol biosynthesis pathway (1182.5)

To cope with hyperosmotic stress cells accumulate compatible organic osmolytes, which replace harmful inorganic electrolytes while maintaining homeostasis of cell volume and intracellular ionic strength. D‐myo‐Inositol (Ins) has been shown to be an important compatible organic osmolyte for fish that are exposed to salinity stress. Two enzymes are required to synthesize Ins: (I) MIPS (Ins phosphate synthase) converts glucose‐6‐phosphate to Ins‐3‐phosphate (Ins‐3‐P), and (II) IMPase (Ins monophosphate phosphatase) dephosphorylates Ins‐3‐P to yield Ins. Both enzymes are induced in different fish species and tissues after exposure to elevated environmental salinity, the result being intracellular accumulation of Ins. We cloned MIPS and IMPase from Mozambique tilapia (Oreochromis mossambicus), expressed the corresponding hexa‐His tagged recombinant proteins in bacteria, and purified recombinant MIPS and IMPase using Ni affinity chromatography. We show that both enzymes are more active at basic pH. In addition, the activity of these tilapia enzymes also depends on known cofactors for mammalian MIPS (NAD+) and IMPase (Mg2+). As is common for many other enzymes, tilapia MIPS activity decreases with increasing osmotic strength in vitro due to an increase in KM at elevated osmolality and a decrease of the kcat/KM ratio. In contrast, IMPase activity increases with elevated osmotic strength, particularly when Ins‐3‐P is used as a substrate. IMPase KM as well as Vmax and kcat all increase when buffer osmolality is elevated. These data suggest that, in addition to the induction of both enzymes at mRNA and protein levels, Ins accumulation during hyperosmotic stress is promoted by direct osmotic effects on IMPase enzymatic activity. Supported by NSF grant IOS‐1049780.