A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose.
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 lambdaZAPII, 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 235 nm, indicating that glycosidic bond cleavage was mediated 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 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.
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
V A
VA
McKie
J P
JP
Vincken
A G
AG
Voragen
L A
LA
van den Broek
E
E
Stimson
H J
HJ
Gilbert
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004274:DNA, Recombinant; D002482:Cellulose; D011133:Polysaccharide-Lyases; C426005:rhamnogalacturonan lyase 11A