Protein disorder: conformational distribution of the flexible linker in a chimeric double cellulase.

The structural properties of the linker peptide connecting the cellulose-binding module to the catalytic module in bimodular cellulases have been investigated by small-angle x-ray scattering. Since the linker and the cellulose-binding module are relatively small and cannot be readily detected separately, the conformation of the linker was studied by means of an artificial fusion protein, Cel6BA, in which an 88-residue linker connects the large catalytic modules of the cellulases Cel6A and Cel6B from Humicola insolens. Our data showed that Cel6BA is very elongated with a maximum dimension of 178 A, but could not be described by a single conformation. Modeling of a series of Cel6BA conformers with interdomain separations ranging between 10 A and 130 A showed that good Guinier and P(r) profile fits were obtained by a weighted average of the scattering curves of all the models where the linker follows a nonrandom distribution, with a preference for the more compact conformers. These structural properties are likely to be essential for the function of the linker as a molecular spring between the two functional modules. Small-angle x-ray scattering therefore provides a unique tool to quantitatively analyze the conformational disorder typical of proteins described as natively unfolded.

Anatomy of glycosynthesis: structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants.

The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).

Structural basis for ligand binding and processivity in cellobiohydrolase Cel6A from Humicola insolens.

The enzymatic digestion of cellulose entails intimate involvement of cellobiohydrolases, whose characteristic active-center tunnel contributes to a processive degradation of the polysaccharide. The cellobiohydrolase Cel6A displays an active site within a tunnel formed by two extended loops, which are known to open and close in response to ligand binding. Here we present five structures of wild-type and mutant forms of Cel6A from Humicola insolens in complex with nonhydrolyzable thio-oligosaccharides, at resolutions from 1.7-1.1 A, dissecting the structural accommodation of a processing substrate chain through the active center during hydrolysis. Movement of ligand is facilitated by extensive solvent-mediated interactions and through flexibility in the hydrophobic surfaces provided by a sheath of tryptophan residues.

Fluorescent cellulose microfibrils as substrate for the detection of cellulase activity.

To devise a sensitive cellulase assay based on substrates having most of the physical characteristics of native cellulose, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was used as a grafting agent to prepare suspensions of fluorescent microfibrils from bacterial cellulose. These suspensions were digested by a series of commercially relevant cellulases from Humicola insolens origin: cloned Cel6B and Cel 45A as well as crude H. insolens complex. The digestion induced the release of fluorescent cellodextrins as well as reducing sugars. After adequate centrifugation, these soluble products were analyzed as a function of grafting content, digestion time, and cellulase characteristics. The resulting data allowed the grafting conditions to be optimized in order to maximize the quantity of soluble products and therefore to increase the sensitivity of the detection. A comparison between the amount of released fluorescence and that of released reducing sugar allowed the differentiation between processive exo and endo cellulase activities. The casting of films of DTAF-grafted microfibrils at the bottom of the microwell titer plates also led to sensitive cellulase detection. As these films kept their integrity and remained firmly glued to the well bottom during the digestion time, they are tailored made for a full automation of the cellulases testing.

Dimension, shape, and conformational flexibility of a two domain fungal cellulase in solution probed by small angle X-ray scattering.

Cellulase Cel45 from Humicola insolens has a modular structure with a catalytic module and a cellulose-binding module (CBM) separated by a 36 amino acid, glycosylated, linker peptide. The solution conformation of the entire two domain Cel45 protein as well as the effect of the length and flexibility of the linker on the spatial arrangement of the constitutive modules were studied by small angle x-ray scattering combined with the known three-dimensional structure of the individual modules. The measured dimensions of the enzyme show that the linker exhibits an extended conformation leading to a maximum extension between the two centers of mass of each module corresponding to about four cellobiose units on a cellulose chain. The glycosylation of the linker is the key factor defining its extended conformation, and a five proline stretch mutation on the linker was found to confer a higher rigidity to the enzyme. Our study shows that the respective positioning of the catalytic module and the CBM onto the insoluble substrate is most likely influenced by the linker structure and flexibility. Our results are consistent with a model where cellulases can move on the surface of cellulose with a caterpillar-like displacement with free energy restrictions.

Chemoenzymatic synthesis of a bifunctionalized cellohexaoside as a specific substrate for the sensitive assay of cellulase by fluorescence quenching.

A new bifunctionalized cellohexaose derivative was synthesized as a specific substrate for continuous assay of cellulases by resonance energy transfer. This cellohexaoside has a naphthalene moiety (EDANS) as a fluorescent energy donor at the reducing end and a 4-(4'-dimethylaminobenzeneazo)-benzene derivative as an acceptor chromophore at the non-reducing end. The key steps for the preparation of the target molecule involved transglycosylation reactions of cellobiosyl and cellotetraosyl fluoride donors onto cellobiosyl acceptors catalysed by the E197A mutant of cellulase Cel7B from Humicola insolens. Upon digestion with various cellulases, the energy transfer was disrupted and an increase of fluorescence was observed.

Crystallization and preliminary X-ray characterization of a thermostable pectate lyase from Thermotoga maritima.

Pectate lyase is an enzyme involved in the degradation of the pectate portion of the primary plant cell wall. A recombinant pectate lyase from Thermotoga maritima where three of the four cysteine residues have been mutated (C132I, C156N, C194L) has been crystallized. Crystals of the same morphology and trigonal space group R3 with similar unit-cell parameters were obtained under two different conditions. The first, 0.3 M (NH(4))H(2)PO(4) pH 4.2, gave crystals with a maximum size of 0.4 x 0.2 x 0.2 mm in one week that diffracted to a resolution of 1.87 A and had unit-cell parameters a = b = 80.6, c = 148.8 A. The second, 0.1 M sodium acetate, 6%(w/v) PEG 4000 pH 6.5, gave the same size crystals in two weeks that diffracted to a resolution of 2.1 A and had unit-cell parameters a = b = 80.0, c = 150.1 A.

Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.

Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.