Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus

The genes glpK ana glpF, encoding glycerol Kinase ana the glycerol facilita tor of Thermus flavus, a member of the Thermus/Deinococcus group, have rece ntly been identified. The protein encoded by glpK exhibited an unusually hi gh degree of sequence identity (80.6 %) when compared to the sequence of gl ycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (64.8 %) was observed when the sequences of the glycerol facilitat ors of the two organisms were compared. The work presented in this paper de monstrates that T. flavus is capable of taking up glycerol, that glpF and g lpK are expressed constitutively and that glucose exerts a repressive effec t on the expression of these genes. T. flavus was found to possess the gene ral components of the phosphoenolpyruvate (PEP):sugar phosphotransferase sy stem (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus glycerol kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-cat alysed phosphorylation in glycerol kinase of Enterococcus casseliflavus. Pu rified glycerol kinase from T. flavus could also be phosphorylated with enz yme I and HPr from B. subtilis. Similar to enterococcal glycerol kinases, p hosphorylated T. flavus glycerol kinase exhibited an electrophoretic mobili ty on denaturing and non-denaturing polyacrylamide gels that is different f rom the electrophoretic mobility of non-phosphorylated glycerol kinase. How ever, in contrast to PEP-dependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphor ylation of T. flavus glycerol kinase caused only a slight increase in enzym e activity.