The Non-catalytic Chitin-binding Protein CBP21 from Serratia marcescens Is Essential for Chitin Degradation

Abstract

The Gram-negative soil bacterium Serratia marcescens uses three different family 18 chitinases to degrade chitin, an abundant insoluble carbohydrate polymer composed of ␤(1,4)-linked units of N-acetylglucosamine. We show that efficient chitin degradation additionally depends on the action of a small noncatalytic protein, CBP21, which binds to the insoluble crystalline substrate, leading to structural changes in the substrate and increased substrate accessibility. CBP21 strongly promoted hydrolysis of crystalline ␤-chitin by chitinases A and C, while it was essential for full degradation by chitinase B. CBP21 variants with single mutations on the largely polar binding surface lost their ability to promote chitin degradation, while retaining considerable affinity for the polymer. Thus, binding alone is not sufficient for CBP21 functionality, which seems to depend on specific, mostly polar interactions between the protein and crystalline chitin. This is the first time a secreted binding protein is shown to assist in the enzymatic degradation of an insoluble carbohydrate via non-hydrolytic disruption of the substrate. Interestingly, homologues of CBP21 occur in most chitin-degrading microorganisms, suggesting a general mechanism by which chitin-binding proteins enhance chitinolytic activity. Homologues also occur in chitinase-containing insect viruses, whose infectiousness is known to depend on chitinase efficiency.Each year more than one billion tons of chitin, a linear polymer of ␤(1-4)-linked N-acetylglucosamine, is produced in the biosphere, mainly by insects, fungi, crustaceans, and other marine organisms (1). Like cellulose, chitin is an abundant insoluble linear polymer with considerable mechanical and chemical strength. Three crystalline forms of chitin have been described in terms of the arrangement of the chitin chains: ␣-chitin (chains run anti-parallel), ␤-chitin (chains run parallel), and ␥-chitin (mixed parallel/antiparallel chains).Despite its resilience, insolubility, and abundant production, chitin does not accumulate in most ecosystems, suggesting that nature has developed efficient processes for chitin degradation.

Chitinase 3-like-1 enhances bacterial adhesion to colonic epithelial cells through the interaction with bacterial chitin-binding protein

“…CHI3L1 has no chitinase activity, 3 but still possesses a binding ability to chitin and chitooligosaccharides with high affinity through a preserved hydrophobic substrate binding cleft, 19,16 and the majority of chitinase-producing pathogenic bacteria have the ability to produce chitin-binding molecules such as CBP21. 22 We found that CBP21 knockout strain of S. marcescens significantly reduced in the adhesion but not invasion rate to CECs. Therefore, CBP21 seems to be involved in the bacterial adhesion to CECs, and CHI3L1 may bind to bacteria through CBPs.…”

“…21 More importantly, the majority of chitinaseproducing pathogenic microorganisms contain a gene encoding for the homolog of the cbp21 gene, suggesting the presence of a potential binding ability of chitinase-producing pathogenic bacteria to chitin via CBP21 homolog. 22 In this study, we propose a new possibility that CHI3L1 expressed on CECs binds to bacterial CBP21 homolog via chitin or chitin-like molecule, and this binding subsequently enhances the adhesion of these bacteria to CECs. To identify the exact mechanism of how CHI3L1 enhances the bacterial adhesion on CECs in vitro, we utilize S. marcescens wild type (WT) in its mutant form and CBP21-overexpressing nonpathogenic E. coli.…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…Thus, CBM33, which is highly expressed in chitin-degrading bacteria such as Serratia marcescens, potentiates the chitinases from this organism, particularly during the latter stages of the degradative process when the glycoside hydrolases are attacking highly crystalline forms of the polysaccharide (Vaaje-Kolstad et al, 2005a). More modest potentiation of cellulases by "noncatalytic" bacterial CBMs has also been reported (Moser et al, 2008), while it has been suggested that several noncatalytic fungal proteins may play a role in plant cell wall disruption.…”

Cleavage of cellulose by a CBM33 protein

“…8,9) and most importantly, these proteins act synergistically with chitinases. 10,11 In a very recent study, it was shown that these proteins in fact are enzymes that cleave chitin chains while still being in their crystalline context, using an unprecedented mechanism that involves a hydrolytic and an oxidative step. 12 Thus, the CBM33 generates two new chain ends on the crystalline surface, one normal nonreducing and an ''oxidized reducing end,'' that is, an aldonic acid.…”