Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module

Abstract

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.

A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose

“…These data support the view that Rgl11A is a rhamnogalacturan-specific lyase that exhibits no activity against polygalacturonic acid. This is in sharp contrast with Pel10A, described in the accompanying paper, which appears to be a classic polygalacturonan lyase, as its primary substrate is polygalacturonic acid [9].…”

“…The data described in this paper and in the accompanying paper [9] demonstrate for the first time that P. cellulosa expresses enzymes that attack pectin. Rgl11A exhibits specificity for substituted rhamnogalacturonans.…”

“…cellulosa, in common with other plant-cell-wall-degrading micro-organisms, contains extensive multigene families encoding cellulases and hemicellulases [28,29]. It is surprising, therefore, that only a single rhamnogalacturonan lyase and pectate lyase [9] were isolated from the Pseudomonas gene library, particularly as plant pathogenic prokaryotes, such as Erwinia, contain extensive multigene families encoding polygalacturonases, pectate lyases and pectin lyases (see http :\\afmb.cnrs-mrs.fr\ "pedro\CAZY\db.html). In addition, our screening strategy did not identify DNA sequences encoding other main-chaincleaving pectinases such as polygalacturonases or rhamnogalacturonan hydrolases.…”

“…In addition, the Pseudomonas enzyme is modular, containing a functional family 2a CBM (CBM2a) module. These data, together with the accompanying paper [9], therefore show that some microbial pectinases contain cellulose-binding domains, and are thus capable of associating with the plant cell wall.…”

“…The C-terminal 90-residue module of Rgl11A is homologous to CBM2a cellulose-binding domains. It contains the five tryptophan residues that are completely conserved in other Pseudomonas CBM2a modules, including pectate lyase 10A (Pel10A) described in the accompanying paper [9], three of which (Trp-794, Trp-831 and Trp-848) have been shown to comprise the cellulose-binding surface of these proteins [26,27]. It is interesting to note that the proposed molecular architecture of Rgl11A is similar to the P. cellulosa pectate lyase described in the accompanying paper ; this enzyme also contains three modules in which the catalytic domain and the CBM2a are at the N-and C-termini, respectively [9].…”