EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain - PubMed (original) (raw)

EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain

M T Rincón et al. Appl Environ Microbiol. 2001 Oct.

Abstract

The mechanisms by which cellulolytic enzymes and enzyme complexes in Ruminococcus spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene endB from Ruminococcus flavefaciens 17 was purified as a His-tagged product after expression in Escherichia coli and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown R. flavefaciens that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945-1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.

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Figures

FIG. 1

Binding of R. flavefaciens 17 endoglucanases to crystalline cellulose (Avicel). Lane 1 shows an SDS-PAGE CMC zymogram of extracellular proteins from an Avicel-grown (150-h) culture of R. flavefaciens 17 that bound to Avicel. Clear zones are the result of CMCase activity, as revealed by Congo red staining. In this case the proteins were from the supernatants obtained from washing harvested cells in buffer (see Materials and Methods), but similar results were obtained with the original culture supernatant (not shown). Lane 2 shows molecular size markers (in kilodaltons), stained with Coomassie blue.

FIG. 2

Diagram showing domain structures of EndB and of the His-tagged derivatives used here. The N-terminal signal peptide is shown in black, the C-terminal dockerin is shown in white, the T-rich region is shown in light gray, the family 44 catalytic domain is indicated by dark cross-hatching, and the unknown region (containing a new CBM) is indicated by light cross-hatching. The primers used for construction of the pET28 clones in order to overexpress the full-length and truncated polypeptides as His6-tagged products are described in the Materials and Methods.

FIG. 3

Multiple alignment of dockerin domains from R. flavefaciens 17 XynD, XynB, EndB and EndA with dockerin from LicB from Clostridium thermocellum (37). Residues conserved in all five sequences are highlighted in black, while those conserved in 4 out of 5 sequences are highlighted in grey. Numbers on the right hand side refer to amino acid sequence positions within the relevant gene product.

FIG. 4

Binding of purified, His-tagged pET-EndB to Avicel. Binding was assayed by incubating the purified EndB protein with Avicel. The Avicel was then washed three times in phosphate buffer, and attached polypeptides were then eluted with SDS sample buffer (see Materials and Methods). CMCase activity was detected by a zymogram technique (36) by incorporating CMC into the gel (lane 1). His-tagged protein was detected by using specific antibodies (see Materials and Methods) (lanes 2 and 3). Incubation with Avicel was at 37°C for 5 min (lanes 1 and 2) or at 4°C for 20 h (lane 3).

FIG. 5

Binding of a purified, His-tagged protein fragment (from pET-EndBΔ N498Δ C106) that carries residues 499 to 702 of EndB to insoluble cellulose (Avicel). The putative EndB CBM was incubated with Avicel for 20 h at 4°C (lanes 1, 2, and 3) or for 1 h at 37°C (lanes 4, 5, and 6), followed by four washes in buffer (see Materials and Methods). Lanes 1 and 4 were loaded with the unbound protein, lanes 2 and 5 were loaded with protein eluted with 2% SDS after binding to Avicel and washing, and lanes 3 and 6 were loaded with the 18-fold-concentrated final buffer wash.

FIG. 6

Binding of purified His-tagged pET-EndB to R. flavefaciens 17 proteins from an Avicel-grown culture. Lane 1, molecular size markers (in kilodaltons); lane 2, R. flavefaciens culture pellet; lane 3, R. flavefaciens culture supernatant; lane 4, R. flavefaciens supernatant proteins after absorption onto Avicel at 37°C, four washes in buffer, and subsequent elution with 2% SDS.

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EndB, a multidomain family 44 cellulase from Ruminococcus flavefaciens 17, binds to cellulose via a novel cellulose-binding module and to another R. flavefaciens protein via a dockerin domain - PubMed (original) (raw)