Abstract
Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.
Highlights
The uptake and concomitant phosphorylation of a wide variety of carbohydrates into bacterial cells is, in many cases, accomplished by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) [1]
The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)(1,10-phenanthroline)3, and they were visualized by SDS-polyacrylamide gel electrophoresis
E. coli containing all four cysteines; SSCS, N-terminal His-tagged Escherichia coli mannitol permease (EIImtl) with Cys110, Cys320, and Cys571 replaced with a serine; SSCSS124C, SSCS with Ser124 in the C domain replaced with a cysteine; IIChis-CL, C-terminal His-tagged cysteine-less C domain with Cys110 and Cys320 replaced with serine; IIChis-S124C, IIChis-CL in which Ser124 is replaced with a cysteine
Summary
The uptake and concomitant phosphorylation of a wide variety of carbohydrates into bacterial cells is, in many cases, accomplished by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) [1]. These kinetic data clearly indicate that SSCS and SSCS-S124C are fully functional enzymes and that both mutant C domains can bind mannitol and form functional heterodimers with EIImtl.
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