Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase

Abstract

In the accompanying paper [Beylot, McKie, Voragen, DoeswijkVoragen and Gilbert (2001) Biochem. J. 358, 607-614] the chromosome of Pseudomonas cellulosa was shown to contain two genes, abf 51A and abf62A, that encode arabinofuranosidases belonging to glycoside hydrolase families 51 and 62, respectively. In this report we show that expression of Abf 51A is induced by arabinose and arabinose-containing polysaccharides. Northernblot analysis showed that abf 51A was efficiently transcribed, whereas no transcript derived from abf62A was detected in the presence of arabinose-containing polysaccharides. Zymogram and Western-blot analyses revealed that Abf 51A was located on the outer membrane of P. cellulosa. To investigate the importance of Abf 51A in the release of arabinose from poly-and oligosaccharides, transposon mutagenesis was used to construct an

Structural and enzymatic characterization of a glycoside hydrolase family 31 α-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification

“…S3), essentially no CjXyl31A was found in the extracellular medium; trace amounts, detectable as a very faint band on the Western blot after prolonged staining, may be attributable to cell lysis late in the exponential growth phase. Localisation of CjXyl31A on the outer membrane is consistent with previous studies on membrane-bound glycosidases of C. japonicus [8,9]. Further, the absence of an aspartic acid residue at position +2 -this residue is a serine in CjXyl31A -is also suggestive of outer membrane localisation [51].…”

“…To delineate the diverse α-glucoside-and α-xyloside-processing activities in GH31, a phylogenetic tree was constructed using only the (β/α) 8 catalytic domain of all members of GH31 whose enzyme specificity has previously been determined (data from the CAZy database [2], www.cazy.org), together with CjXyl31A (Fig. 2).…”

“…The central catalytic domain forms an (β/α) 8 -barrel consisting of residues 412-791 and with two significant "inserts" beyond the standard barrel termed insert region-1 (Ins1) at residue 512-556 and insert region-2 (Ins2) at residue 586-613. The (β/α) 8 -barrel contains the catalytic apparatus, as will be described subsequently in light of a covalent intermediate complex and by virtue of similarities to related GH31 and Clan GH-D enzymes of known 3-D structure. The N-terminal region clearly forms two essentially discrete lobes.…”

“…To date, the three dimensional structure has been determined for five members of GH31, with YicI from Escherichia coli being the only α-xylosidase [29]. These five enzymes all display a highly similar fold, with a central catalytic (α/β) 8 In the case of CjXyl31A, substrate specificity has two facets: (1) this strictly exo-acting enzyme is specific for a terminal, unsubstituted xyloside and (2) this xyloside must lie at the extreme reducing end of a xylogluco-oligosaccharide; there is no activity on the internal xylosides of substrates such as XXXG and XXXGXXXG. The specificity for an unmodified terminal xylose moiety is provided, as with other GH31 enzymes, by the constellation of residues that interact directly with the -1 subsite xyloside.…”

“…In contrast with anaerobic species, such as the Clostridia, the enzymes of C. japonicus that target polysaccharides integral to the plant cell walls do not form large membrane-attached cellulosome complexes [7], but are instead either secreted into the medium or attached to the cell membrane as lipoproteins. Indeed, about one third of the encoded carbohydrate-degrading enzymes have been predicted to be lipoproteins [6], which may suggest that C. japonicus has developed a strategy involving the ultimate degradation of plant cell wall-derived oligosaccharides in close proximity to the cell, as has been shown, for example, in the case of xylan utilisation [8,9].…”