Properties of a Cyclodextrin-specific, Unusual Porin from Klebsiella oxytoca

Abstract

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 peptidecontaining precursor, and possesses a high content of antiparallel ␤-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 ␣-cyclodextrin. Titration experiments allowed the calculation of a half-saturation constant, K S , of 28 M 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, ␥ ‫؍‬ 93°. 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.

Chitoporin from Vibrio harveyi, a Channel with Exceptional Sugar Specificity

“…Our study revealed that chitohexaose is the most potent substrate of the VhChiP channel, as it blocked the ion flow even at nanomolar concentrations, and the monomeric subunit was already saturated below 1 M. In Table 2, we summarize the rate constants: the on-rate (k on ) is by far the highest for chitohexaose, whereas the off-rate (k off ) is the lowest. Consequently, the resultant binding constant (K) of 5.0 ϫ 10 6 M Ϫ1 is 1-5 orders stronger than the reported values for other analogs (25,31,(35)(36)(37)(38)(39)(40). According to the kinetic data in Table 2, VhChiP is the most active sugar-specific channel reported to date.…”

Outer-membrane translocation of bulky small molecules by passive diffusion

“…In the absence of the cymA gene, growth on CDs is lost (4). In addition, CymA confers to an E. coli ΔlamB strain the ability to use maltopentaose and maltohexaose for growth (5). These data indicate that CymA functions as an efficient OM uptake channel for α-and β-CD as well as for linear maltooligosaccharides.…”

Reversible Pore Block of Connexin Channels by Cyclodextrins

“…Several bacterial proteins bind or transport CDs (CymE, CymA, and CycB) (20,22,95,96). Their affinities for CDs range from 30 M to 3 mM depending on the protein and the CD.…”

“…In this context, CDs have been referred to as "molecular adapters" (21). In contrast, CDs appear to fully block the ionic conductance of CymA pores (20,22).…”

“…CDs have been shown to reversibly enter and modify the pore properties of ␣-hemolysin (␣HL) (19) and CymA bacterial porin (20). When lodged in the ␣HL pore, CDs reduce the ionic conductance.…”