Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIAB(Man) subunit of the mannose transporter of Escherichia coli.
The hydrophilic subunit of the mannose transporter (IIAB(Man)) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N-terminal domain (Trp12, Trp33, and Trp69) and one in the C-terminal domain (Trp182). Single and double Trp-Phe mutants of IIABMan and of the IIA domain were produced. Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment. Stabilities of the Trp mutants of dimeric IIA(Man) and IIAB(Man) were estimated from midpoints of the GdmHCl-induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively. W12F exhibited increased stability, but only 6% of the wild-type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active. Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F. These results suggest that flexibility is required for the adjustments of protein-protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIA(Man), IIB(Man), and the incoming mannose bound to the transmembrane IIC(Man)-IID(Man) complex.
Markovic-Housley
Z
Z
Department of Structural Biology, Biocenter, University of Basel, Switzerland.
Stolz
B
B
Lanz
R
R
Erni
B
B
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D010649:Phenylalanine / Q000737:chemistry*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry*
D010649:Phenylalanine; D014364:Tryptophan; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C037992:phosphoenolpyruvate-mannose phosphotransferase