celA, another gene coding for a multidomain cellulase from the extreme thermophile Caldocellum saccharolyticum

Abstract

Caldocellum saccharolyticum is an extremely thermophilic anaerobic bacterium capable of growth on cellulose and hemicellulose as sole carbon sources. Cellulase and hemicellulase genes have been found clustered together on its genome. The gene for one of the cellulases (celA) was isolated on a lambda genomic library clone, sequenced and found to comprise a large open-reading frame of 5253 base pairs that could be translated into a peptide of 1751 amino acids. To date, it is the largest cellulase gene sequenced. The translated product is a multidomain structure composed of two catalytic domains and two cellulose-binding domains linked by proline-threonine-rich regions (PT linkers). The N-terminal domain of celA encodes for an endoglucanase activity on carboxymethylcellulose, consistent with its high homology to the sequences of several other endo-1, 4-beta-D-glucanases. The carboxyterminal domain shows sequence homology with a cellulase from Clostridium thermocellum (CelS), which is known to act synergistically with a second component to hydrolyze crystalline cellulose. In the absence of a Caldocellum homologue for this second protein, we can detect no activity from this domain.

Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp

“…Examination of the sequence to the 3Ј end of the celB gene revealed the beginning of an open reading frame which potentially coded for another cellulase. This open reading frame (celC) extended beyond the 3Ј end of the genomic DNA insert of NZP2 and contained regions of strong homology to the beginning of manA (17) and to the endoglucanase coding region of celA (36). Because of the proximity of the celC gene to the celA, manA, and celB genes, it was predicted that celC would encode yet another multidomain, bifunctional cellulase-hemicellulase of similar overall organization to the CelA, CelB, and ManA enzymes.…”

“…NZP2 is a recombinant lambda 1059 bacteriophage containing approximately 12 kb of C. saccharolyticus genomic DNA which codes for cellulase, xylanase, and mannanase activities. The isolation of NZP2 from a C. saccharolyticus Sau3A genomic library and the identification, subcloning, and sequencing of the celA, manA, and celB genes from NZP2 have been described previously (17,21,23,31,36).…”

“…The last three genes compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb genomic fragment contained within the recombinant lambda bacteriophage NZP2. The celA, celB, and manA genes encode novel, bifunctional enzymes composed of two distinct catalytic domains linked to cellulose-binding domains by proline-and threonine-rich linkers (4,17,31,36). Unlike most other cellulases (18), the CelA, CelB, and ManA cellulose-binding domains are positioned centrally between the two flanking catalytic domains.…”

Thermophilic lignocellulose deconstruction

“…Large, multimodular carbohydrate‐active enzymes were discovered to be the distinguishing feature of the genus Caldicellulosiruptor , with CelB (Table ) being the first described modular multifunctional enzyme (Saul et al ., ), followed by the sequencing of other modular enzymes, such as manA (Gibbs et al ., ) and celA (Te'O et al ., ). Incidentally, although discovered almost 20 years ago, CelA remains one of the largest discrete cellulase proteins known (1751 amino acids, predicted molecular weight of 194.8 kDa).…”

“…Sequenced genomic library clones from C. saccharolyticus (Te'O et al ., ) and Caldicellulosiruptor sp. Tok7B.1 (Bergquist et al ., ; Gibbs et al ., ) were the first observations of co‐located, large modular enzymes containing CBM3s (Fig.…”