Abstract:
Chitin, a biopolymer of β-1,4-glycosidic linked GlcNAc residues, is an abundant source of
carbon and nitrogen for microorganisms. Chitin degradation by microorganism is
achieved using a complex pathway including various enzymes and transporters. Diffusion
of chitin oligosaccharides across the outer membrane of Gram-negative bacteria usually
occurs through chitoporin channels. In this study we described the characterization of a
novel chitoporin (so-called EcChiP) which helps to uptake chitin oligosaccharides in nonchitinolytic E. coli. Single channel recoding in Black Lipid Membrane (BLM) reconstitution
technique demonstrated that EcChiP form monomeric channel with specificity towards
chitooligosaccharides. The molecular mass obtained by size exclusion chromatography for
the purified EcChiP was 60,000 Da, which is in good agreement with theoretical molecular
weight for monomeric channel. Together with bulk permeation study by liposome
swelling assays, we demonstrate that EcChiP is a sugar-specific transporter, with
pronounced specificity towards long-chain chitooligosaccharides such as chitohexaose,
chitopentaose and chitotetraose. Thermodynamic assessment by isothermal titration
microcalorimetry (ITC) suggested that chitohexaose-EcChiP channel interactions are
driven by an endothermic process, yielding a binding constant (K) value of 2.5 x105 M-1.
Analysis of protein fluorescence enhancement suggested that the binding process was
hydrophobic. Non-linear curve fitting of the chitohexaose titration curve from protein
fluorescence enhancement yielded the binding constant (K) of 2.9x105 M-1, a value that
agreed well with the values obtained from ITC experiments and from single channel
recordings. For the first time, these data provide insights into chitooligosaccharide uptake
by OccD-like chitoporin in non-chitinolytic bacteria.
