Novel substrate specificity engineered in the arabinose binding protein

The L-arabinose binding protein (ABP) of Escherichia coli naturallybinds L-arabinose and D-galactose with very high affinity and,with reduced affinity, a variety of other sugars that differonly at the C5 position of the pyranose ring.However, thereare stringent specificity requirements at the 1, 2, 3 and 4positions. Based on the high resolution crystallographic structureof the Ugand-protein complex, remodelling of the binding pocketwas attempted to shift the specificity towards Cl-substitutedgalactosides. To create space in the vicinity of the reducingend of bound galactose, four residues, LyslO, Asp90, Thrl47and Leul45, have been mutated for residues with smaller sidechains. Forty-seven mutants containing different combinationsof these mutations were tested by fluorometry for their abilityto bind methylß-D-galactoside (met-ß-Gal)or iso-propyl-ß-D-thio-galactoside (IPTG). Two double-residuemutants carrying Ser at position 147 and Ala or Gly at position90 appeared of particular interest for being able to bind met-ß-Galor IPTG, respectively, and no longer galactose. Fluorescenceexperiments and molecular modelling indicate that the mode ofbinding of the new substrates to the mutant proteins might besimilar to that of the natural ligands to wild-type ABP.