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Modification of catalytically important carboxy residues in endoglucanase D from Clostridium thermocellum.

ABSTRACT: Endoglucanase D (EC 3.2.1.4; EGD) from Clostridium thermocellum is rapidly (k = 216 M-1.min-1) and almost completely (greater than 95%) inactivated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulphonate). Spectrophotometric analysis at 340 nm reveals that eight carboxy residues react, whereas specific ligands protect one residue against modification. The enzyme retains it full activity under the latter conditions. The kinetics and pH-dependence of inactivation point towards the involvement of one or more essential carboxy groups with a pKa of 5.7-5.8. Samples modified in the absence or presence of ligand were analysed by reversed-phase liquid chromatography after proteolysis with subtilisin. Dual-wavelength monitoring at 214 and 340 nm during this fractionation leads to the identification of a putatively active-site peptide (Gly-508-Ala-562) which was further characterized by amino acid and partial N-terminal sequence analyses. Asp-546 and Glu-555 are postulated as possible active-site residues. This follows from alignments using ten endoglucanase sequences belonging to the same family. Strong local conservation suggests that this C-terminal sequence is structurally and/or functionally important.

SUBMITTER: Tomme P

PROVIDER: S-EPMC1132783 | biostudies-other | 1992 Jul

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA. RESULTS:Although all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity. CONCLUSIONS:The identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future.