Novel substrate specificity engineered in the arabinose binding protein.
The L-arabinose binding protein (ABP) of Escherichia coli naturally binds L-arabinose and D-galactose with very high affinity and, with reduced affinity, a variety of other sugars that differ only at the C5 position of the pyranose ring. However, there are stringent specificity requirements at the 1, 2, 3 and 4 positions. Based on the high resolution crystallographic structure of the ligand-protein complex, remodelling of the binding pocket was attempted to shift the specificity towards C1-substituted galactosides. To create space in the vicinity of the reducing end of bound galactose, four residues, Lys10, Asp90, Thr147 and Leu145, have been mutated for residues with smaller side chains. Forty-seven mutants containing different combinations of these mutations were tested by fluorometry for their ability to bind methyl-beta-D-galactoside (met-beta-Gal) or iso-propyl-beta-D-thio-galactoside (IPTG). Two double-residue mutants carrying Ser at position 147 and Ala or Gly at position 90 appeared of particular interest for being able to bind met-beta-Gal or IPTG, respectively, and no longer galactose. Fluorescence experiments and molecular modelling indicate that the mode of binding of the new substrates to the mutant proteins might be similar to that of the natural ligands to wild-type ABP.
Declerck
N
N
Laboratoire de Génétique, Institut National Agronomique Paris-Grignon, France.
Abelson
J
J
D000595:Amino Acid Sequence; D001089:Arabinose / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D008756:Methylgalactosides / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
C016242:AraF protein, E coli; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008756:Methylgalactosides; C031552:methyl beta-galactoside; D007544:Isopropyl Thiogalactoside; D001089:Arabinose; D005690:Galactose