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.
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April 2001
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Research Article|
February 26 2001
Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module
Ian E. BROWN;
Ian E. BROWN
*School of Sciences, University of Sunderland, Sunderland SR1 3SD, U.K.
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Marie H. MALLEN;
Marie H. MALLEN
1
*School of Sciences, University of Sunderland, Sunderland SR1 3SD, U.K.
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Simon J. CHARNOCK;
Simon J. CHARNOCK
†Department of Chemistry, University of York, York YO1 5DD, U.K.
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Gideon J. DAVIES;
Gideon J. DAVIES
†Department of Chemistry, University of York, York YO1 5DD, U.K.
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Gary W. BLACK
*School of Sciences, University of Sunderland, Sunderland SR1 3SD, U.K.
3To whom correspondence should be addressed (e-mail gary.black@unn.ac.uk).
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Biochem J (2001) 355 (1): 155–165.
Article history
Received:
July 13 2000
Revision Received:
November 22 2000
Accepted:
January 12 2001
Citation
Ian E. BROWN, Marie H. MALLEN, Simon J. CHARNOCK, Gideon J. DAVIES, Gary W. BLACK; Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module. Biochem J 1 April 2001; 355 (1): 155–165. doi: https://doi.org/10.1042/bj3550155
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