New Family of Carbohydrate-Binding Modules Defined by a Galactosyl-Binding Protein Module from a Cellvibrio japonicus Endo-Xyloglucanase

Abstract

Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, for example, by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is a valuable source for CAZyme and CBM discovery and characterization due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (X181), and a glycoside hydrolase family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase (M. A. Attia, C. E. Nelson, W. A. Offen, N. Jain, et al., Biotechnol Biofuels 11:45, 2018, https://doi.org/10.1186/s13068-018-1039-6). In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest products of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes. IMPORTANCE This study reveals carbohydrate-binding module family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the carbohydrate-active enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

Keywords: Bacteroidetes; Cellvibrio; Gammaproteobacteria; carbohydrate-active enzyme; carbohydrate-binding modules (CBMs); glycoside hydrolase; mannan; plant biomass; polysaccharide; xyloglucan; xyloglucanase.

FIG 1

Modular architecture of the native C. japonicus CJA_2959 gene product and different constructs used in the study. (A) The full-length gene product is composed of a signal peptide, an Fn3 domain, an X181 module, and a catalytic GH5_4 (CjGH5F) domain. (B) Recombinant proteins produced for characterization using the E. coli expression vector pET28a and the ligation-independent cloning (LIC) vectors pMCSG53, pMCSG69, and pMCSG-GST-TEV (67). MBP, GST, and sfGFP are connected to X181 or Fn3-X181 by a TEV cleavage site.

FIG 2

Binding capacity of sfGFP, Fn3-X181-sfGFP, and sfGFP-CjCBM2 for Avicel. Recombinant sfGFP (27.7 kDa; lanes 1 and 2), His₆-Fn3-X181-sfGFP (48.7 kDa; lanes 3 and 4), and sfGFP-CjCBM2 (40.7 kDa; lanes 5 and 6) were incubated with Avicel, and unbound proteins were removed by centrifugation (lanes 1, 3, 5, and 7). Bound proteins were released from Avicel by boiling in SDS-PAGE loading dye (lanes 2, 4, 6, and 8). sfGFP was used as a negative control, while sfGFP-CjCBM2 (31) was used as a positive control.

FIG 3

Affinity gel electrophoresis for Fn3-X181-sfGFP against different polysaccharide substrates. Recombinant protein bands were visualized by fluorescence (A) and Coomassie blue staining (B) following electrophoresis at 90 V for 10 h. The negative-control sfGFP completely migrated through under these conditions. (C) Gel following 5.5 h of electrophoresis, which demonstrates no binding of the negative-control sfGFP toward tamarind galactoxyloglucan (fluorescence visualization). BBG, barley β-glucan; HEC, hydroxyethyl cellulose; KGM, konjac glucomannan; XyG, tamarind galactoxyloglucan; GalMan, galactomannan.

FIG 4

Isothermal titration calorimetry of Fn3-X181-sfGFP against different ligands. (A) Tamarind seed xyloglucan. (B) Glc₄-based tamarind seed xyloglucan oligosaccharides. The top graph in each pair shows the raw heat during titration, while the bottom graph shows the integrated heat after correction.