Cellulase Cel5A from alkalophilic Bacillus sp. 1139 contains a family 17 carbohydrate-binding module (BspCBM17) and a family 28 CBM (BspCBM28) in tandem. The two modules have significantly similar amino acid sequences, but amino acid residues essential for binding are not conserved. BspCBM28 was obtained as a discrete polypeptide by engineering the cel5A gene. BspCBM17 could not be obtained as a discrete polypeptide, so a family 17 CBM from endoglucanase Cel5A of Clostridium cellulovorans, CcCBM17, was used to compare the binding characteristics of the two families of CBM. Both CcCBM17 and BspCBM28 recognized two classes of binding sites on amorphous cellulose: a high affinity site (K a ϳ1 ؋ 10 6 M ؊1 ) and a low affinity site (K a ϳ2 ؋ 10 4 M ؊1 ). They did not compete for binding to the high affinity sites, suggesting that they bound at different sites on the cellulose. A polypeptide, BspCBM17/ CBM28, comprising the tandem CBMs from Cel5A, bound to amorphous cellulose with a significantly higher affinity than the sum of the affinities of CcCBM17 and BspCBM28, indicating cooperativity between the linked CBMs. Cel5A mutants were constructed that were defective in one or both of the CBMs. The mutants differed from the wild-type enzyme in the amounts and sizes of the soluble products produced from amorphous cellulose. This suggests that either the CBMs can modify the action of the catalytic module of Cel5A or that they target the enzyme to areas of the cellulose that differ in susceptibility to hydrolysis.

In the commonly accepted mechanism for enzymatic hydrolysis of cellulose, endo-beta-1,4-glucanases randomly cleave glucosidic bonds within glucan polymers, providing sites for attack by exo-cellobiohydrolases (EC 3.2.1.91). It has been proposed that hydrolysis by Trichoderma reesei cellobiohydrolase II is restricted to the ends of cellulose polymers because two surface loops cover its active site to form a tunnel. In a closely related endoglucanase, E2 from Thermomonospora fusca, access to the substrate appears to be relatively unhindered because the carboxyl-proximal loop is shortened, and the amino-proximal loop is displaced. The hypothesis was examined by deletion of a region in Cellulomonas fimi cellobiohydrolase A corresponding to part of the carboxyl-proximal loop of T. reesei cellobiohydrolase II. The mutation enhanced the endoglucanase activity of the enzyme on soluble O-(carboxymethyl)cellulose and altered its activities on 2',4'-dinitrophenyl-beta-D-cellobioside, insoluble cellulose, and cellotetraose.

The cenC gene, encoding ␤-1,4-glucanase C (CenC) from Cellulomonas fimi, was overexpressed in Escherichia coli with a tac-based expression vector. The resulting polypeptide, with an apparent molecular mass of 130 kDa, was purified from the cell extracts by affinity chromatography on cellulose followed by anion-exchange chromatography. N-terminal sequence analysis showed the enzyme to be properly processed. Mature CenC was optimally active at pH 5.0 and 45؇C. The enzyme was extremely active on soluble, fluorophoric, and chromophoric glycosides (4-methylumbelliferyl ␤-glycosides, 2-chloro-4-nitrophenyl-␤-D-cellobioside, and 2-chloro-4-nitrophenyl-lactoside) and efficiently hydrolyzed carboxymethyl cellulose, barley ␤-glucan, lichenan, and, to a lesser extent, glucomannan. CenC also hydrolyzed acid-swollen cellulose, Avicel, and bacterial microcrystalline cellulose. However, degradation of the latter was slow compared with its degradation by CenB, another C. fimi cellulase belonging to the same enzyme family. CenC acted with inversion of configuration at the anomeric carbon, in accordance with its classification as a family 9 member. The enzyme released mainly cellobiose from soluble cellodextrins and insoluble cellulose. Attack appeared to be from the reducing chain ends. Analysis of carboxymethyl cellulose hydrolysis suggests that CenC is a semiprocessive enzyme with both endo-and exoglucanase activities.

The gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA). Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85,349 Da. It is composed of five domains: an N-terminal catalytic domain, three repeated sequences of 95 amino acids, and a C-terminal cellulose-binding domain typical of other C. fimi glycanases. The structure and enzymatic activities of the CbhA catalytic domain are closely related to those of CBH II, an exocellobiohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesei. CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family.

We have characterized by NMR spectroscopy the three active site (His80, His85, and His205) and two non-active site (His107 and His114) histidines in the 34 kDa catalytic domain of Cellulomonas fimi xylanase Cex in its apo, noncovalently aza-sugar-inhibited, and trapped glycosyl-enzyme intermediate states. Due to protection from hydrogen exchange, the level of which increased upon inhibition, the labile 1Hdelta1 and 1H epsilon1 atoms of four histidines (t1/2 approximately 0.1-300 s at 30 degrees C and pH approximately 7), as well as the nitrogen-bonded protons in the xylobio-imidazole and -isofagomine inhibitors, could be observed with chemical shifts between 10.2 and 17.6 ppm. The histidine pKa values and neutral tautomeric forms were determined from their pH-dependent 13C epsilon1-1H epsilon1 chemical shifts, combined with multiple-bond 1H delta2/epsilon1-15N delta1/epsilon2 scalar coupling patterns. Remarkably, these pKa values span more than 8 log units such that at the pH optimum of approximately 6 for Cex activity, His107 and His205 are positively charged (pKa > 10.4), His85 is neutral (pKa < 2.8), and both His80 (pKa = 7.9) and His114 (pKa = 8.1) are titrating between charged and neutral states. Furthermore, upon formation of the glycosyl-enzyme intermediate, the pKa value of His80 drops from 7.9 to <2.8, becoming neutral and accepting a hydrogen bond from an exocyclic oxygen of the bound sugar moiety. Changes in the pH-dependent activity of Cex due to mutation of His80 to an alanine confirm the importance of this interaction. The diverse ionization behaviors of the histidine residues are discussed in terms of their structural and functional roles in this model glycoside hydrolase.