Biochemical properties of glycerol kinase from the hypersaline-adapted archaeon Haloferax volcanii

Extremophilic microorganisms hold promise to serve as robust biocatalysts in the conversion of glycerol waste into high value products. Haloferax volcanii is a hypersaline-adapted archaeon that prefers glycerol over glucose and channels this carbon source into central metabolism through glycerol kinase (GK). Here we report the biochemical properties of the H. volcanii GK and evaluated its potential for biotechnological applications. The N-terminal His-tagged form of GK was found functional in vivo and was readily purified to homogeneity at 4.5-fold higher yield (3 mg/L culture) than GK fused to a C-terminal StrepII tag. Further analysis of His-GK by size exclusion chromatography revealed the enzyme exhibited a glycerol-induced shift from a homodimer to a homodimer-homotetramer equilibrium. Purified His-GK demonstrated robust activity over a broad pH and salinity range, with optimal activity at 100 mM NaCl and 50-60 {degrees}C. The enzyme was catalytically active in organic solvent (5-10 % DMSO) and crude glycerol containing methanol. His-GK was also found to exhibit full activity after freeze-thaw, showed prolonged thermotolerance in 2 M NaCl supplemented buffers, and had a melting temperature (Tm) in the range of 83-84 {degrees}C. Kinetic analysis using the Hill equation indicated His-GK displayed positive cooperativity for glycerol, ATP, and magnesium, with manganese and cobalt also found to serve as divalent cation cofactors. These findings underscore the unique and robust enzymatic properties of H. volcanii GK, representing the first known GK to exhibit positive cooperativity with glycerol and ATP, and highlighting its potential for biotechnological applications in glycerol conversion. IMPORTANCEThis study provides new insight into glycerol kinase kinetics through the discovery of an enzyme exhibiting positive cooperativity for glycerol and ATP. By purifying and characterizing H. volcanii glycerol kinase, this work marks the first isolation of a glycerol kinase from a halophilic archaeon. The enzyme displays unique properties, including resilience to organic solvents, high temperatures, and extreme salinity. It also exhibits sigmoidal kinetics, with Hill coefficients averaging n = 2 for glycerol, ATP, and magnesium, indicating positive cooperativity. This behavior, previously unobserved in other glycerol kinases, aligns with the preference of H. volcanii for glycerol over glucose. Given the role of glycerol kinases in converting glycerol waste into value-added products, their efficiency is often a bottleneck in bacterial systems that prefer glucose and struggle under extreme conditions. The distinctive properties of H. volcanii glycerol kinase suggest potential for biotechnological applications in harsh environments.