Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides

Abstract

Many enzymes that hydrolyze insoluble crystalline polysaccharides such as cellulose and chitin guide detached single-polymer chains through long and deep active-site clefts, leading to processive (stepwise) degradation of the polysaccharide. We have studied the links between enzyme efficiency and processivity by analyzing the effects of mutating aromatic residues in the substrate-binding groove of a processive chitobiohydrolase, chitinase B from Serratia marcescens. Mutation of two tryptophan residues (Trp-97 and Trp-220) close to the catalytic center (subsites +1 and +2) led to reduced processivity and a reduced ability to degrade crystalline chitin, suggesting that these two properties are linked. Most remarkably, the loss of processivity in the W97A mutant was accompanied by a 29-fold increase in the degradation rate for single-polymer chains as present in the soluble chitin-derivative chitosan. The properties of the W220A mutant showed a similar trend, although mutational effects were less dramatic. Processivity is thought to contribute to the degradation of crystalline polysaccharides because detached single-polymer chains are kept from reassociating with the solid material. The present results show that this processivity comes at a large cost in terms of enzyme speed. Thus, in some cases, it might be better to focus strategies for enzymatic depolymerization of polysaccharide biomass on improving substrate accessibility for nonprocessive enzymes rather than on improving the properties of processive enzymes.

Fig. 1.

Enzyme–substrate interactions in ChiB. (A) Surface representation showing aromatic side chains lining the substrate-binding cleft and the binding surface of the chitin-binding domain (extending to the right; residues 479 and 481). The catalytic Glu-144 is colored green. (B) Surface representation of the E144Q mutant in complex with chitopentaose [(GlcNAc)₅] bound to subsites −2 to +3 (27), showing that the substrate-binding cleft has a closed “roof” when substrate is bound. (GlcNAc)₅ is shown with a yellow van der Waals surface. The surfaces of aromatic residues in the protein are blue. (C) Stereo picture showing (GlcNAc)₅ and aromatic residues near the catalytic center. Individual sugars in the pentamer are labeled by the number of the enzyme subsite (from −2 to +3) to which they bind.

Fig. 2.

Schematic picture of ChiB in complex with a single chitin chain. The enzyme has six subsites, numbered −3 to +3. CBM, carbohydrate-binding module (8). The reducing end sugar is colored gray. A correctly positioned N-acetyl group (symbolized by small black balls on sticks) in the −1 subsite is essential for catalysis (which is “substrate-assisted”) to occur (–27). Initial binding of the substrate will produce an odd- or even-numbered “overhang” leading to an odd- or even-numbered product (a trimer or dimer in case of exo activity). The scheme shows the situation during subsequent processive action when only dimers are produced. The arrow indicates the direction of the sliding of the substrate through the active-site cleft (31, 32). In the case of chitosan, complexes formed during processive action may be nonproductive because the sugar bound in the −1 subsite may lack the N-acetyl group. This leads to the production of longer even-numbered oligomers that is diagnostic for processivity (ref. ; see text).

Fig. 3.

Characteristics of wild-type ChiB and the W97A mutant. (A and B) Size exclusion chromatography of products formed during degradation of chitosan (65% acetylated water-soluble chitin with random distribution of acetylated units) with ChiB (A) and W97A (B), after cleavage of 14% of the glycosidic bonds (i.e., α = 0.14). The products are labeled by chain length or, for the shortest products, by sequence (A, acetylated unit; D, deacetylated unit). (C and D) Degradation of chitohexaose with ChiB (C) and W97A (D). ▿, (GlcNAc)₆; □, (GlcNAc)₄; ▵, (GlcNAc)₃; ○, (GlcNAc)₂. (E) Degradation of chitin with ChiB (▴) and W97A (■). (F) Time curve for chitosan degradation with ChiB (▴) and W97A (■). α, fraction of cleaved glycosidic bonds (complete conversion of the substrate to exclusively dimers would yield α = 0.50).