Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus.
The genes glpK and glpF, encoding glycerol kinase and the glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (80-6%) when compared to the sequence of glycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (64.8%) was observed when the sequences of the glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (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-catalysed phosphorylation in glycerol kinase of Enterococcus casseliflavus. Purified glycerol kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal glycerol kinases, phosphorylated T. flavus glycerol kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated glycerol kinase. However, in contrast to PEP-dependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphorylation of T. flavus glycerol kinase caused only a slight increase in enzyme activity.
Darbon
Emmanuelle
E
Laboratoire de Génétique des Microorganismes, INRA-CNRS URA 1925, F-78850 Thiverval-Grignon, France1.
Ito
Kiyoshi
K
School of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852, Japan2.
Huang
Hua-Shan
HS
School of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852, Japan2.
Yoshimoto
Tadashi
T
School of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852, Japan2.
Poncet
Sandrine
S
Laboratoire de Génétique des Microorganismes, INRA-CNRS URA 1925, F-78850 Thiverval-Grignon, France1.
Deutscher
Josef
J
Laboratoire de Génétique des Microorganismes, INRA-CNRS URA 1925, F-78850 Thiverval-Grignon, France1.
D000595:Amino Acid Sequence; D001345:Autoradiography; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000378:metabolism; D005991:Glycerol Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D016415:Sequence Alignment; D013824:Thermus / Q000201:enzymology*
D001426:Bacterial Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D005991:Glycerol Kinase; D005990:Glycerol