Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by Thermobifida fusca Cel9A

Abstract

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(؊1) O1. R378, a residue interacting with Glc(؉1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(؊2) to Glc(؊4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(؊2) to Glc(؊4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

“…Our results suggest that a CBM3c can indeed bind to insoluble cellulose although the binding appeared weak. A sequence alignment of CbCel9B/ Man5A with its homologs revealed that considerable differences exist in the amino acid residues proposed to interact with the ligand (29,32,33) between CbCel9B/Man5A CBM3c with its homologues. Notably, the Q553, R557, E559, and R563 residues in ThefuCel9A, proposed to interact with the ligand, are replaced by E545, K549, Q561, and K565, respectively, in the CBM3c of CbCel9B/Man5A (see Fig.…”

Advantages of a distant cellulase catalytic base

“…Active Site Homology -To better understand why the TrCel6A active site is tuned to require a wire water, we compared its active site to that of another inverting glycoside hydrolase, Tf Cel9A, a processive endocellulase that is believed to employ the classical mechanism based on crystallography and site-directed mutagenesis studies (26,27). We created a Tf Cel9A model based on a product-state crystal structure (PDB ID: 4TF4 (28)) to compare with a product-state structure produced for TrCel6A in the course of our previous work (16), as shown in Figure 7.…”

“…The distances between the glycosidic oxygens and catalytic bases, as well as the presence of two active site waters in structures apparently primed for hydrolysis, indicate that members of this family generally perform hydrolysis via a Grotthuss mechanism to a more distant catalytic base. Our simulations clearly indicate why a longer catalytic base decreases activity; however, the question remains as to why the active site is not tuned for a longer side chain, or why the aspartate is not positioned closer to the cleavage site.Active Site Homology -To better understand why the TrCel6A active site is tuned to require a wire water, we compared its active site to that of another inverting glycoside hydrolase, Tf Cel9A, a processive endocellulase that is believed to employ the classical mechanism based on crystallography and site-directed mutagenesis studies (26,27). We created a Tf Cel9A model based on a product-state crystal structure (PDB ID: 4TF4 (28)) to compare with a product-state structure produced for TrCel6A in the course of our previous work (16), as shown in Figure 7.…”

Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus

“…An extensive hydrogen-bonding network observed between the oligosaccharide substrate and the substrate binding and catalytic residues of the glycosidic enzymes indicates the substrate length might be a determinant in the overall glycosidic enzyme rate (Li et al, 2007;Zhou et al, 2004). Hrmova et al (1998) reported an increased catalytic efficiency with increasing degree of polymerization in a GH1 family BGL from barley.…”