Development of an overexpression system for production of recombinant Thermobifida fusca cellulase Cel6A and its multidimensional NMR analysis

Cellulosic material, including discarded cellulosic waste, can be converted to useful chemicals and energy by employing the cellulolytic machinery of cellulose degrading organisms. Structure-function knowledge of cellulolytic enzymes, as well as optimizing the production of these enzymes, can be used to improve performance in biotechnological applications. Due to its thermostability and broad pH optimum, the Thermomobifida fusca endocellulase Cel6A has promise for economical use in biotechnology. The structure of the 30.4 kD, containing 286 amino acids, Cel6A catalytic domain (cd) has been previously solved by X-ray crystallography. In the crystal structure a conserved aspartic acid residue, D79, resides in a loop 11 Å away from the site of substrate cleavage. In this study direct evidence for the movement of this loop during catalysis into a position closer to the site of substrate cleavage, as suggested by site directed mutagenesis studies, was sought by nuclear magnetic resonance (NMR) spectroscopy: NMR spectroscopy offers the ability to look at proteins in their natural environment allowing conformational changes to be visualized. A monomeric high activity mutant Cel6A E263D catalytic domain (cd) was studied.; In order to perform multidimensional NMR experiments, protein samples with incorporated 2H, 15N, and 13C isotopes were required. Due to the high cost of suitable growth medium and poorly characterized metabolic pathways limiting labeling, the Streptomyces lividans expression system, used prior to this project to produce recombinant T. fusca cellulases, was abandoned. An optimized recombinant protein expression system, producing high yields of isotopically labeled, correctly folded, active, and soluble Cel6A E263D cd protein, was developed in Escherichia coli. The well-characterized metabolic pathways and ability to grow on minimal medium made E. coli a superior candidate for producing labeled Cel6A protein samples for NMR. An optimal expression strain was constructed, followed by optimization of various growth conditions, an essential step for ensuring high yields. The resulting E. coli production system allows easy, efficient, and inexpensive production and purification of labeled Cel6A E263D cd protein samples for multidimensional NMR as well as for other applications.; Movement of the D79 loop upon substrate binding in the active site of the Cel6A cellulase was investigated by titration with a substrate analogue followed by backbone assignments of the free and the substrate-bound enzyme. Changes in several chemical shifts were detected over the titration indicating changes in chemical environment of the involved residues introduced by the substrate. The backbone chemical shifts were analyzed from a set of 2D and 3D spectra. Due to inconsistencies in the detected chemical shifts only 201 backbone cross-shifts were assignable. No chemical shift could be assigned for D79 indicating that the residue may indeed move, and at a speed that renders it non-detectable in the timescale of the NMR experiments.