Chitin, an insoluble polymer of N-acetylglucosamine, is one of the most abundant biopolymers on Earth. By degrading chitin, chitinolytic bacteria such as Vibrio harveyi are critical for chitin recycling and maintenance of carbon and nitrogen cycles in the world’s oceans. A decisive step in chitin degradation is the uptake of chito-oligosaccharides by an outer membrane protein channel named chitoporin (ChiP). Here, we report X-ray crystal structures of ChiP from V. harveyi in the presence and absence of chito-oligosaccharides. Structures without bound sugar reveal a trimeric assembly with an unprecedented closing of the transport pore by the N-terminus of a neighboring subunit. Substrate binding ejects the pore plug to open the transport channel. Together with molecular dynamics simulations, electrophysiology and in vitro transport assays our data provide an explanation for the exceptional affinity of ChiP for chito-oligosaccharides and point to an important role of the N-terminal gate in substrate transport.

Chitin is the most abundant biopolymer in marine ecosystems. However, there is no accumulation of chitin in the ocean-floor sediments, since marine bacteria Vibrios are mainly responsible for a rapid turnover of chitin biomaterials. The catabolic pathway of chitin by Vibrios is a multi-step process that involves chitin attachment and degradation, followed by chitooligosaccharide uptake across the bacterial membranes, and catabolism of the transport products to fructose-6-phosphate, acetate and NH₃. This study reports the isolation of the gene corresponding to an outer membrane chitoporin from the genome of Vibrio harveyi. This porin, expressed in E. coli, (so called VhChiP) was found to be a SDS-resistant, heat-sensitive trimer. Immunoblotting using anti-ChiP polyclonal antibody confirmed the expression of the recombinant ChiP, as well as endogenous expression of the native protein in the V. harveyi cells. The specific function of VhChiP was investigated using planar lipid membrane reconstitution technique. VhChiP nicely inserted into artificial membranes and formed stable, trimeric channels with average single conductance of 1.8±0.13 nS. Single channel recordings at microsecond-time resolution resolved translocation of chitooligosaccharides, with the greatest rate being observed for chitohexaose. Liposome swelling assays showed no permeation of other oligosaccharides, including maltose, sucrose, maltopentaose, maltohexaose and raffinose, indicating that VhChiP is a highly-specific channel for chitooligosaccharides. We provide the first evidence that chitoporin from V. harveyi is a chitooligosaccharide specific channel. The results obtained from this study help to establish the fundamental role of VhChiP in the chitin catabolic cascade as the molecular gateway that Vibrios employ for chitooligosaccharide uptake for energy production.

Chitoporin (VhChiP) is a sugar-specific channel responsible for the transport of chitooligosaccharides through the outer membrane of the marine bacterium Vibrio harveyi. Single channel reconstitution into black lipid membrane allowed single chitosugar binding events in the channel to be resolved. VhChiP has an exceptionally high substrate affinity, with a binding constant of K = 5.0 × 10⁶ m⁻¹ for its best substrate (chitohexaose). The on-rates of chitosugars depend on applied voltages, as well as the side of the sugar addition, clearly indicating the inherent asymmetry of the VhChiP lumen. The binding affinity of VhChiP for chitohexaose is 1–5 orders of magnitude larger than that of other known sugar-specific porins for their preferred substrates. Thus, VhChiP is the most potent sugar-specific channel reported to date, with its high efficiency presumably reflecting the need for the bacterium to take up chitin-containing nutrients promptly under turbulent aquatic conditions to exploit them efficiently as its sole source of energy.

Since chitin is a highly abundant natural biopolymer, many attempts have been made to convert this insoluble polysaccharide into commercially valuable products using chitinases and β-N-acetylglucosaminidases (GlcNAcases). We have previously reported the structure and function of chitinase A from Vibrio harveyi 650. This study t reports the identification of two GlcNAcases from the same organism and their detailed functional characterization.

Four crystal structures of human YKL-39 were solved in the absence and presence of chitooligosaccharides. The structure of YKL-39 comprises a major (β/α)8 TIM barrel domain and a small α+β insertion domain. Structural analysis demonstrates that YKL-39 interacts with chitooligosaccharides through hydrogen bonds and hydrophobic interactions. The binding of chitin fragments induces local conformational changes that facilitate tight binding. Compared with other GH-18 members, YKL-39 has the least extended chitin-binding cleft, containing five subsites for sugars, namely (-3)(-2)(-1)(+1)(+2), with Trp360 playing a prominent role in the sugar-protein interactions at the centre of the chitin-binding cleft. Evaluation of binding affinities obtained from isothermal titration calorimetry and intrinsic fluorescence spectroscopy suggests that YKL-39 binds to chitooligosaccharides with Kd values in the micromolar concentration range and that the binding energies increase with the chain length. There were no significant differences between the Kd values of chitopentaose and chitohexaose, supporting the structural evidence for the five-binding subsite topology. Thermodynamic analysis indicates that binding of chitooligosaccharide to YKL-39 is mainly driven by enthalpy.