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

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Abstract
The enzyme IImannitol (EIImtl) 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 IIImtl 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 EIImtl 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 EIImtl 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 EIImtl of E. coli [Pas, H. H. & Robillard, G. T. (1988) Biochemistry 27, 5835–5839], is well conserved in EIImtl 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.