Mannitol‐specific enzyme II of the phosphoenolpyruvate‐dependent phosphotransferase system of Staphylococcus carnosus

Abstract

The enzyme II^mannitol (EII^mtl) of the phosphoenolpyruvate‐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 NH₂‐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.