Glycosynthase-mediated assembly of xylanase substrates and inhibitors.

An exo-ß-xylosidase mutant with glycosynthase activity was created to aid in the synthesis of xylanase substrates and inhibitors. Simple monosaccharides were easily elaborated into di-, tri- and tetrasaccharides by using this enzyme. Some products proved to be surprisingly potent inhibitors of xylanases from glycoside hydrolase families 10 and 11.

Probing electrostatic interactions along the reaction pathway of a glycoside hydrolase: histidine characterization by NMR spectroscopy.

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.

NMR spectroscopic characterization of a beta-(1,4)-glycosidase along its reaction pathway: stabilization upon formation of the glycosyl-enzyme intermediate.

NMR spectroscopy was used to search for mechanistically significant differences between the thermodynamic and dynamic properties of the 34 kDa (alpha/beta)8-barrel catalytic domain of beta-(1,4)-glycosidase Cex (or CfXyn10A) in its free (apo-CexCD) and trapped glycosyl-enzyme intermediate (2FCb-CexCD) states. The main chain chemical shift perturbations due to the covalent modification of CexCD with the mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside are limited to residues within its active site. Thus, consistent with previous crystallographic studies, formation of the glycosyl-enzyme intermediate leads to only localized structural changes. Furthermore, 15N relaxation methods demonstrated that the backbone amide and tryptophan side chains of apo-CexCD are very well ordered on both the nanosecond to picosecond and millisecond to microsecond time scales and that these dynamic features also do not change significantly upon formation of the trapped intermediate. However, covalent modification of CexCD led to the increased protection of many amides and indoles, clustered around the active site of the enzyme, against fluctuations leading to hydrogen exchange. Similarly, thermal denaturation studies demonstrated that 2FCb-CexCD has a significantly higher midpoint unfolding temperature than apo-CexCD. The covalently modified protein also exhibited markedly increased resistance to proteolytic degradation by thermolysin relative to apo-CexCD. Thus, the local and global stability of CexCD increase along its reaction pathway upon formation of the glycosyl-enzyme intermediate, while its structure and fast time scale dynamics remain relatively unperturbed. This may reflect thermodynamically favorable interactions with the relatively rigid active site of Cex necessary to bind, distort, and subsequently hydrolyze glycoside substrates.

N-Glycosidase-carbohydrate-binding module fusion proteins as immobilized enzymes for protein deglycosylation.

A carbohydrate-binding module (CBM) was fused to the N-termini of mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (EndoF1) and peptide N-glycosidase F (PNGaseF), two glycosidases from Chryseobacterium meningosepticum that are used to remove N-linked glycans from glycoproteins. The fusion proteins CBM-EndoF1 and CBM-PNGaseF also carry a hexahistidine tag for purification by immobilized metal affinity chromatography after production by Escherichia coli. CBM-EndoF1 is as effective as native EndoF1 at deglycosylating RNaseB; the glycans released by both enzymes are identical. Like native PNGaseF, CBM-PNGaseF is active on denatured but not on native RNaseB. Both fusion proteins are as active on RNaseB when immobilized on cellulose as they are in solution. They retain activity in the immobilized state for at least 1 month at 4 degrees C. The hexahistidine tag can be removed with thrombin, leaving the CBM as the only affinity tag. The CBM can be removed with factor Xa if required.

Production of a self-activating CBM-factor X fusion protein in a stable transformed Sf9 insect cell line using high cell density perfusion culture.

Factor Xa is a serine protease, whose high selectivity can be used to cleave protein tags from recombinant proteins. A fusion protein comprised of a self-activating form of factor X linked to a cellulose-binding module, saCBMFX, was produced in a stable transformed Sf9 insect cell line. The activity of the insect cell produced saCBMFX was higher than the equivalent mammalian cell produced material. A 1.5 l batch fermentation reached a maximum cell concentration of 1.6 x 10(7) cells ml(-1) and a final saCBMFX concentration of 4 mg l(-1). The production of saCBMFX by this cell line was also analyzed in a 1.5 l perfusion system using an ultrasonic filter as a cell-retention device for flow rates up to 3.5 l day(-1). The cell-retention efficiency of an air backflush mode of acoustic filter operation was greater than 95% and eliminated the need to pump the relatively shear sensitive insect cells. In the perfusion system over 4 x 10(7) Sf9 cells ml(-1) were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 x 10(7) cells ml(-1) the saCBMFX production rate was doubled to 6 mg l(-1) day(-1). The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l(-1) day(-1)) by an order of magnitude.