Substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae.
We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-beta,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.
Tavoulari
Sotiria
S
Laboratory of Biological Chemistry, University of Ioannina Medical School, Ioannina GR-45110, Greece.
Frillingos
Stathis
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D016972:Enterobacter cloacae / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry* / Q000235:genetics
D026901:Membrane Transport Proteins; D027981:Symporters; C021548:melibiose permease; C021499:lactose permease