Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module.
Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5 kDa. 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 9.2 kDa 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 14.2 kDa 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 35.8 kDa 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 pH 10 and in the presence of 2 mM Ca2+. Highest enzyme activity was detected at 62 degrees 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.
School of Sciences, University of Sunderland, Sunderland SR1 3SD, U.K.
I E
IE
Brown
M H
MH
Mallen
S J
SJ
Charnock
G J
GJ
Davies
G W
GW
Black
D000595:Amino Acid Sequence; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology*; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D010368:Pectins; D002482:Cellulose; D011133:Polysaccharide-Lyases; C426004:pectate lyase 10A; C003181:polygalacturonic acid