Properties of a cyclodextrin-specific, unusual porin from Klebsiella oxytoca.
The function of CymA, 1 of the 10 gene products involved in cyclodextrin uptake and metabolism by Klebsiella oxytoca, was characterized. CymA is essential for growth on cyclodextrins, but it can also complement the deficiency of a lamB (maltoporin) mutant of Escherichia coli for growth on linear maltodextrins, indicating that both cyclic and linear oligosaccharides are accepted as substrates. CymA was overproduced in E. coli and purified to apparent homogeneity. CymA is a component of the outer membrane, is processed from a signal peptide-containing precursor, and possesses a high content of antiparallel beta-sheet. Incorporation of CymA into lipid bilayers and conductance measurements revealed that it forms ion-permeable channels, which exhibit a substantial current noise. CymA-induced membrane conductance decreased considerably upon addition of alpha-cyclodextrin. Titration experiments allowed the calculation of a half-saturation constant, K(S), of 28 microM for its binding to CymA. CymA assembled in vitro to two-dimensionally crystalline tubular membranes, which, on electron microscopy, are characterized by a p1-related two-sided plane group. The crystallographic unit cell contains four monomeric CymA molecules showing a central pore. The lattice parameters are a = 16.1 nm, b = 3.8 nm, gamma = 93 degrees. CymA does not form trimeric complexes in lipid membranes and shows no tendency to trimerize in solution. CymA thus is an atypical porin with novel properties specialized to transfer cyclodextrins across the outer membrane.
Pajatsch
M
M
Institute of Genetics and Microbiology, University of Munich, Maria-Ward-Strasse 1a, D-80638 Munich, Germany.
Andersen
C
C
Mathes
A
A
Böck
A
A
Benz
R
R
Engelhardt
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002455:Cell Division; D003505:Cyclodextrins / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D007709:Klebsiella / Q000737:chemistry*; D008051:Lipid Bilayers / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000648:ultrastructure; D018272:Porins / Q000737:chemistry* / Q000648:ultrastructure; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017421:Sequence Analysis; D017550:Spectroscopy, Fourier Transform Infrared; D047391:alpha-Cyclodextrins
D001426:Bacterial Proteins; D003505:Cyclodextrins; D008051:Lipid Bilayers; D018272:Porins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D047391:alpha-Cyclodextrins; C105942:CymA protein, Pseudomonas aureofaciens; D010088:Oxidoreductases; C032613:alpha-cyclodextrin