A new family of rhamnogalacturonan lyases contains an enzyme that binds tocellulose

Pseudomonas cellulosa is an aerobic bacterium that synthesizes an extensive array of modular cellulases and hemicellulases, which have a modular archi tecture consisting of catalytic domains and distinct non-catalytic carbohyd rate-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 lambda ZAPII was screened for pec tinase-encoding sequences. A recombinant phage that attacked arabinan, gala ctan and rhamnogalacturonan was isolated. The encoded enzyme, designated Rg l11A, had a modular structure comprising an N-terminal domain that exhibite d homology to Bacillus and Streptomyces proteins of unknown function. a mid dle 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 i ts C-terminal module was a functional cellulose-binding domain, and the N-t erminal module consisted of a catalytic domain that hydrolysed rhamnogalact uronan-containing substrates. The activity of Rgl11A against apple- and pot ato-derived rhamnogalacturonan substrates indicated that the enzyme had a s trong preference for rhamnogalacturonans that contained galactose side chai ns, and which were not esterified. The enzyme had an absolute requirement f or calcium, a high optimum pH, and catalysis was associated with an increas e in absorbance at 235 nm, indicating that glycosidic bond cleavage was med iated via a beta -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 Bl ack (2001) Biochem. J, 355, 155-165] suggests that the capacity to bind cel lulose plays an important role in the activity of main-chain-cleaving Pseud omonas pectinases, in addition to cellulases and hemicellulases.