R. Antony J. Warren

Characterization and affinity applications of cellulose-binding domains

Cellulose-binding domains (CBDs) are discrete protein modules found in a large number of carbohydrolases and a few nonhydrolytic proteins. To date, almost 200 sequences can be classified in 13 different families with distinctly different properties. CBDs vary in size from 4 to 20 kDa and occur at different positions within the polypeptides; N-terminal, C-terminal and internal. They have a moderately high and specific affinity for insoluble or soluble cellulosics with dissociation constants in the low micromolar range. Some CBDs bind irreversibly to cellulose and can be used for applications involving immobilization, others bind reversibly and are more useful for separations and purifications. Dependent on the CBD used, desorption from the matrix can be promoted under various different conditions including denaturants (urea, high pH), water, or specific competitive ligands (e.g. cellobiose). Family I and IV CBDs bind reversibly to cellulose in contrast to family II and III CBDs which are in general, irreversibly bound. The binding of family II CBDs (CBD(Cex)) to crystalline cellulose is characterized by a large favourable increase in entropy indicating that dehydration of the sorbent and the protein are the major driving forces for binding. In contrast, binding of family IV CBDs (CBD(N1)) to amorphous or soluble cellulosics is driven by a favourable change in enthalpy which is partially offset by an unfavourable entropy change. Hydrogen bond formation and van der Waals interactions are the main driving forces for binding. CBDs with affinity for crystalline cellulose are useful tags for classical column affinity chromatography. The affinity of CBD(N1) for soluble cellulosics makes it suitable for use in large-scale aqueous two-phase affinity partitioning systems.

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

The Agrobacterium sp. β-glucosidase (Abg) is a retaining β-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using α-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using α-d-galactopyranosyl fluoride and 4-nitrophenyl β-d-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.

The cellulose‐binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding

Cellulomonas fimi endo‐β‐1, 4‐glucanase A (CenA) contains a discrete N‐terminal cellulose‐binding domain (CBDcenA)‐ Related CBDs occur In at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDcenA‐ The adsorption of CBDcenA to Crystalline cellulose was compared with that of two Trp mutants (W14A and W68A). The affinities of the mutant CBDs for cellulose were reduced by approximately 50‐ and 30‐fold, respectively, relative to the wild type. Physical measurements indicated that the mutant CBDs fold normally. Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface.

The E358S mutant of Agrobacterium sp. β‐glucosidase is a greatly improved glycosynthase

Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizing oligosaccharides. The ‘original’ glycosynthase, derived from Agrobacterium sp. β‐glucosidase (Abg) by mutating the nucleophile glutamate to alanine (E358A), synthesizes oligosaccharides in yields exceeding 90% [Mackenzie, L.F., Wang, Q., Warren, R.A.J. and Withers, S.G. (1998) J. Am. Chem. Soc. 120, 5583–5584]. This mutant has now been re‐cloned with a His6‐tag into a pET‐29b(+) vector, allowing gram scale production and single step chromatographic purification. A dramatic, 24‐fold, improvement in synthetic rates has also been achieved by substituting the nucleophile with serine, resulting in improved product yields, reduced reaction times and an enhanced synthetic repertoire. Thus poor acceptors for Abg E358A, such as PNP‐GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP‐β‐LacNAc. The increased glycosylation activity of Abg E358S likely originates from a stabilizing interaction between the Ser hydroxyl group and the departing anomeric fluorine of the α‐glycosyl fluoride.

β-Mannosynthase: Synthesis of β-Mannosides with a Mutant β- Mannosidase

Engineering enzymes: The glutamic acid nucleophile of a retaining β-mannosidase has been replaced with a serine residue to form a β-mannosynthase. When the new enzyme is provided with an α-mannosyl fluoride donor and an appropriate acceptor, β-mannoside linkages are synthesized. Remarkably, α-mannosyl fluoride can be generated in situ by providing the mannosynthase with excess fluoride ion.

Fusion to an endoglucanase allows alkaline phosphatase to bind to cellulose

Endoglucanase CenA of Cellulomonas fimi comprises an N‐terminal cellulose‐binding domain and a C‐terminal catalytic domain joined together by a sequence of 23 proline and threonine residues (the Pro‐Thr box). The domains function independently when separated by proteolysis. TnphoA has been used to generate cenA′‐′phoA fusions. CenA′‐′PhoA fusion polypeptides which contain the entire cellulose‐binding domain of CenA bind to cellulose, allowing their purification from periplasmic extracts in a single, facile step. This result has implications for purification or immobilisation of chimeric proteins on a cheap cellulose matrix.

Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a β‐1,4‐exoceilobiohydrolase analogous to Trichoderma reesei CBH II

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 cataiytic domain are closely related to those of CBH ll, an exocelloblohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesel. 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.

Co‐operative binding of triplicate carbohydrate‐binding modules from a thermophilic xylanase

Family 6 carbohydrate‐binding modules were am‐plified by polymerase chain reaction (PCR) from Clostridium stercorarium strain NCIB11754 genomic DNA as a triplet. Individually, these modules bound to xylooligosaccharides and cellooligosaccharides with affinities varying from ≈ 3 × 103 M–1 to ≈ 1 × 105 M–1. Tandem and triplet combinations of these modules bound co‐operatively to soluble xylan and insoluble cellulose to give ≈ 20‐ to ≈ 40‐fold increases in affinity relative to the individual modules. This co‐operativity was an avidity effect resulting from the modules within the tandems and triplet interacting simul‐ taneously with proximal binding sites on the polysac‐charides. This occurred by both intrachain and interchain interactions. The duplication or triplication of modules appears to be linked to the growth temperature of the organism; co‐operativity in these multiplets may compensate for the loss of affinity at higher temperatures.

Affinity electrophoresis for the identification and characterization of soluble sugar binding by carbohydrate-binding modules

Affinity electrophoresis was used to identify and quantify the interaction of carbohydrate-binding modules (CBMs) with soluble polysaccharides. Association constants determined by AE were in excellent agreement with values obtained by isothermal titration calorimetry and fluorescence titration. The method was adapted to the identification, study and characterization of mutant carbohydrate-binding modules with altered affinities and specificities. Competition affinity electrophoresis was used to monitor binding of small, soluble mono- and disaccharides to one of the modules.

Expansion of the glycosynthase repertoire to produce defined manno-oligosaccharides

Mutant endo-mannanases, in which the catalytic nucleophile has been replaced, function as glycosynthases in the synthesis of manno-oligosaccharides of defined lengths.