Biochem. J. (2001) 355
(167–177) (Printed in Great Britain)
A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose
Vincent A. MCKIE*, Jean-Paul VINCKEN†, Alphons G. J. VORAGEN†, Lambertus A. M. VAN DEN BROEK†, Elaine STIMSON‡ and Harry J. GILBERT*1
*Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K., †Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands, and ‡DuPont (U.K.) Ltd, 40 Station Road, Cambridge CB1 2UJ, U.K.
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 lZAPII, 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 b-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.
Key words: carbohydrate-binding module, polysaccharide lyase family 11.
Abbreviations used: CBM, carbohydrate-binding module; CBM2a, family 2a CBM; Fn3, fibronectin type 3 module; HPAEC, high-performance anion-exchange chromatography; HPSEC, high-performance size-exclusion chromatography; MHR-S, saponified modified hairy region; Pel10A, pectate lyase 10A; Rgl11A, rhamnogalacturonan lyase 11A; RDA, Red-Dyed Arabinan; GST, glutathione S-transferase; Caps, 3-(cyclohexylamino)propane-1-sulphonic acid; ORF, open reading frame; PSP, putative secreted protein.
1To whom correspondence should be addressed (e-mail H.J.Gilbert@Newcastle.ac.uk).
The nucleotide sequence depicted in Figure 3 has been submitted to the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AY026755.
Received 13 July 2000/22 November 2000; accepted 12 January 2001
The Biochemical Society, London ©
2001
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