A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose

• Published in: Biochemical journal 2001-04, Vol.355 (1), p.167-177
• Main Authors: MCKIE, Vincent A., VINCKEN, Jean-Paul, VORAGEN, Alphons G. J., VAN DEN BROEK, Lambertus A. M., STIMSON, Elaine, GILBERT, Harry J.
• Format: Article
• Language: English
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3550167

Pseudomonas cellulosa is an aerobic bacterium that synthesizes an extensive array of modular cellulases and hemicellulases, which have a modular architecture consisting of catalytic domains and distinct non-catalytic carbohydrate-binding modules (CBMs). To investigate whether the main-chain-cleaving pectinases from this bacterium also have a modular structure, a library of P. cellulosa genomic DNA, constructed in λZAPII, was screened for pectinase-encoding sequences. A recombinant phage that attacked arabinan, galactan and rhamnogalacturonan was isolated. The encoded enzyme, designated Rgl11A, had a modular structure comprising an N-terminal domain that exhibited homology to Bacillus and Streptomyces proteins of unknown function, a middle domain that exhibited sequence identity to fibronectin-3 domains, and a C-terminal domain that was homologous to family 2a CBMs. Expression of the three modules of the Pseudomonas protein in Escherichia coli showed that its C-terminal module was a functional cellulose-binding domain, and the N-terminal module consisted of a catalytic domain that hydrolysed rhamnogalacturonan-containing substrates. The activity of Rgl11A against apple- and potato-derived rhamnogalacturonan substrates indicated that the enzyme had a strong preference for rhamnogalacturonans that contained galactose side chains, and which were not esterified. The enzyme had an absolute requirement for calcium, a high optimum pH, and catalysis was associated with an increase in absorbance at 235nm, indicating that glycosidic bond cleavage was mediated via a β-elimination mechanism. These data indicate that Rgl11A is a rhamnogalacturonan lyase and, together with the homologous Bacillus and Streptomyces proteins, comprise a new family of polysaccharide lyases. The presence of a family 2a CBM in Rgl11A, and in a P. cellulosa pectate lyase described in the accompanying paper [Brown, Mallen, Charnock, Davies and Black (2001) Biochem. J. 355, 155–165] suggests that the capacity to bind cellulose plays an important role in the activity of main-chain-cleaving Pseudomonas pectinases, in addition to cellulases and hemicellulases.