Expression, purification and characterization of the enzyme II mannitol-specific domain from Staphylococcus carnosus and determination of the active-site cysteine residue.
The C-terminal B domain of mannitol-specific enzyme II (enzyme IIB) of the phosphoenolpyruvate-dependent phosphotransferase system for mannitol from Staphylococcus carnosus was subcloned, purified and characterized. In Staphylococcal cells, mannitol-specific enzyme II is composed of a soluble A domain (EIIA) and a transmembrane C domain transporter with a fused enzyme IIB (IIB) domain. We purified large amounts of the IIB domain as an in-frame fusion with six histidine residues. Here, we show that the domain is stable and can be phosphorylated by phosphoenolpyruvate and the phosphotransferase components. It is a dimer over a wide range of pH values and salt conditions. Differences between the published nucleotide sequence data and the mass-spectroscopic data obtained with the purified protein lead to anewed nucleotide sequencing of the gene. Two errors in the original proposed sequence were found, the correction of the second error leading to a frame shift that adds 10 amino acids to the deduced amino acid sequence. The mass of the phosphorylated domain is 20,068 Da, 80 Da more than the mass of the unphosphorylated domain, therefore, no other residues, such as COOH side chains, are directly involved in an additional phosphate linkage concerning the IIB domain. 31P-NMR experiments as well as chemical modification proved that Cys429 is the phosphoamino acid. Titration of the phosphorylated domain during 31P-NMR did not lead to the typical shift for the protonation of the thiophosphate in the resonance spectrum. Thus, the thiophosphate remains in the twofold negatively charged state.
Pogge von Strandmann
R
R
Arbeitsgruppe Physiologie der Mikroorganismen, Abteilung Biologie, Ruhr-Universität Bochum, Germany.
Weigt
C
C
Fischer
R
R
Meyer
H E
HE
Kalbitzer
H R
HR
Hengstenberg
W
W
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010766:Phosphorylation; D011487:Protein Conformation; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics*
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D003545:Cysteine