Reduced Na ⁺ Affinity Increases Turnover of Salmonella enterica Serovar Typhimurium MelB

Abstract

؉ , Li؉ , or H ؉ . Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na ؉ -binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (V max ) for melibiose transport in Escherichia coli DW2 and greatly decrease Na ؉ affinity, indicating that intracellular release of Na ؉ is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop 4-5 and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases V max with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.

Structure-based mechanism for Na+/melibiose symport by MelB

“…It is noteworthy that a short cytoplasmic stretch of helix V (Arg141-Arg149) links the sugar-binding site with two ionic locks (L-1 and L-2) (Fig. 6a, b) and also contains two other conformationally important residues, Glu142 42 and Pro148 47 . We propose that sugar binding promotes a dynamic state of the cytoplasmic stretch of helix V (Fig.…”

“…After transfer onto the PVDF membrane, MelB St proteins were detected with anti-His tag antibody, and imaged by the ImageQuant TM LAS 4000 Biomolecular Imager (GE Health Care Life Science) 47 .…”

Effect of Detergents on Galactoside Binding by Melibiose Permeases

“…6, 13, 24 Briefly, stepwise addition of melibiose was performed during D 2 G FRET until no further change in the FRET signal was observed. The IC 50 was determined by hyperbolic fitting (OriginPro).…”

Survival of Escherichia coli Cells on Solid Copper Surfaces Is Increased by Glutathione

“…Due to the similarities in charge and ionic diameter, Cu I may be transported instead of Na I by sodium-dependent transport systems such as MelB (53,54) (Fig. 2).…”