Open AccessMannitol‐specific enzyme II of the phospho enol pyruvate‐dependent phosphotransferase system of Staphylococcus carnosus
Author(s) -
FISCHER Roland,
HENGSTENBERG Wolfgang
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb16717.x
Subject(s) - pep group translocation , biochemistry , biology , escherichia coli , operon , peptide sequence , nucleic acid sequence , enzyme , microbiology and biotechnology , amino acid , structural gene , cysteine , phosphoenolpyruvate carboxykinase , gene
The enzyme II mannitol (EII mtl ) of the phospho enol pyruvate‐dependent phosphotransferase system (PTS) catalyses the uptake and concomitant phosphorylation of mannitol by bacteria; it is specified by the gene mtlA. MtlA is located near the genes mtlF and mtlD in the staphylococcal genome, encoding the enzyme III mtl and the mannitol‐1‐phosphate dehydrogenase, respectively. We present the cloning of the whole operon by a novel complementation system which is generally suitable for cloning Gram‐positive PTS genes. The nucleotide sequence of a 2.5‐kbp subclone spanning mtlA has been determined. From the deduced amino acid sequence, it is predicted that the membrane‐protein EII mtl consists of 505 amino acid residues (54112Da). The protein has the expected hydropathy profile of an integral‐membrane protein. The NH 2 ‐terminal part of the enzyme resides within the membrane, whereas the COOH‐terminus of the enzyme has the properties of a soluble protein. Comparison with the known amino acid sequence of EII mtl of Escherichia coli [Lee, C. A. & Saier, M. H. (1983) J. Biol. Chem. 258 , 10 761–10 767] showed significant similarity. The motif containing the cysteine, which is the putative second phosphorylation site in EII mtl of E. coli [Pas, H. H. & Robillard, G. T. (1988) Biochemistry 27 , 5835–5839], is well conserved in EII mtl of Staphylococcus carnosus . Chemical modification of the single active site cysteine residue by Ellman's reagent leads to total inactivation, which can be reversed by treatment with 2‐mercaptoethanol.