Biochem. J. (2001) 355
(155–165) (Printed in Great Britain)
Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module
Ian E. BROWN*, Marie H. MALLEN*1, Simon J. CHARNOCK†, Gideon J. DAVIES† and Gary W. BLACK*2,3
*School of Sciences, University of Sunderland, Sunderland SR1 3SD, U.K., and †Department of Chemistry, University of York, York YO1 5DD, U.K.
Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5kDa. Sequence analysis revealed that Pel10A contained a signal peptide and two serine-rich linker sequences that separate three modules. Sequence similarity was seen between the 9.2kDa N-terminal module of Pel10A and family 2a carbohydrate-binding modules (CBMs). This N-terminal module of Pel10A was shown to encode an independently functional module with affinity to crystalline cellulose. A high sequence identity of 66% was seen between the 14.2kDa central module of Pel10A and the functionally uncharacterized central modules of the xylan-degrading enzymes endoxylanase 10B, arabinofuranosidase 62C and esterase 1D, also from P. cellulosa. The 35.8kDa C-terminal module of Pel10A was shown to have 30 and 36% identities with the family 10 pectate lyases from Azospirillum irakense and an alkaliphilic strain of Bacillus sp. strain KSM-P15, respectively. This His-tagged C-terminal module of the Pel10A was shown to encode an independent catalytic module (Pel10Acm). Pel10Acm was shown to cleave pectate and pectin in an endo-fashion and to have optimal activity at pH10 and in the presence of 2mM Ca2+. Highest enzyme activity was detected at 62°C. Pel10Acm was shown to be most active against pectate (i.e. polygalacturonic acid) with progressively less activity against 31, 67 and 89% esterified citrus pectins. These data suggest that Pel10A has a preference for sequences of non-esterified galacturonic acid residues. Significantly, Pel10A and the P. cellulosa rhamnogalacturonan lyase 11A, in the accompanying article [McKie, Vincken, Voragen, van den Broek, Stimson and Gilbert (2001) Biochem. J. 355, 167–177], are the first CBM-containing pectinases described to date.
Key words: b-elimination, esterified pectin, homogalacturonan, pectinase, rhamnogalacturonan.
Abbreviations used: CE, carbohydrate esterase; CBM, carbohydrate-binding module; CDTA, cyclohexane-trans-1,2-diaminetetra-acetate; ECP, esterified citrus pectin; GH, glycosyl hydrolase; Pel10A, pectate lyase 10A; Pel10Acm, catalytic module of Pel10A; Pel10Acbm2a, family 2a CBM of Pel10A; PGA, polygalacturonic acid; Rgl11A, rhamnogalacturonan lyase 11A; PL, polysaccharide lyase; Caps, 3-(cyclohexylamino)propane-1-sulphonic acid; LB, Luria–Bertani; HPAEC, high-performance anion-exchange chromatography.
1Present address: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
2Present address: School of Applied and Molecular Sciences, University of Northumbria at Newcastle, Newcastle upon Tyne NE1 8ST, U.K.
3To whom correspondence should be addressed (e-mail gary.black@unn.ac.uk).
The nucleotide sequence of the pel10A gene has been submitted to the GenBank Nucleotide Sequence Database under accession number AF279264.
Received 13 July 2000/22 November 2000; accepted 12 January 2001
The Biochemical Society, London ©
2001
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