Unusual sequence organization in CenB, an inverting endoglucanase from Cellulomonas fimi

Abstract

The nucleotide sequence of the cenB gene was determined and used to deduce the amino acid sequence of endoglucanase B (CenB) of Cellulomonasfimi. CenB comprises 1,012 amino acids and has a molecular weight of 105,905. The polypeptide is divided by so-called linker sequences rich in proline and hydroxyamino acids into five domains: a catalytic domain of 607 amino acids at the N terminus, followed by three repeats of 98 amino acids each which are >60% identical, and a C-terminal domain of 101 amino acids which is 50% identical to the cellulose-binding domains of C. fimi cellulases Cex and CenA. A deletion mutant of the cenB gene encodes a polypeptide lacking the C-terminal 333 amino acids of CenB. The truncated polypeptide is catalytically active and, like intact CenB, binds to cellulose, suggesting that CenB has a second cellulosebinding site. The sequence of amino acids 1 to 461 of CenB is 35% identical, with a further 15% similarity, to that of a cellulase from avocado, which places CenB in cellulase family E. CenB releases mostly cellobiose and cellotetraose from cellohexaose. Like CenA, CenB hydrolyzes the ,3-1,4-glucosidic bond with inversion of the anomeric configuration. The pH optimum for CenB is 8.5, and that for CenA is 7.5.

Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-1,4-glucanase

“…The gene encodes a polypeptide of 747 amino acids. The codon usage of the cenD gene is very similar to that of cex, cenA, cenB, and cenC (8,28,33,42 (Fig. 3).…”

“…A third oligodeoxynucleotide (1; see Fig. 4) was synthesized which corresponded to the six N-terminal amino acids of mature CenD, using the codon bias of other sequenced C. fimi genes (8,28,33,42). The combination of oligodeoxynucleotides 1 and 3 allowed amplification of an 800-bp fragment of C fimi DNA by PCR.…”

“…Although it hydrolyzes carboxymethyl cellulose (CM-cellulose), Cex is properly classified as a xylanase (11). All of these enzymes contain discrete cellulose-binding domains (CBDs) which can function independently of the catalytic domains (7,8,15,24,28). While the CBDs of CenA, CenB, and Cex are closely related, the two N-terminal CBDs of CenC are only distantly related (7).…”

“…While the CBDs of CenA, CenB, and Cex are closely related, the two N-terminal CBDs of CenC are only distantly related (7). CenA and Cex (44 and 47 kDa, respectively) both comprise a catalytic domain joined to a CBD by a short linker polypeptide; CenB and CenC (106 and 113 kDa, respectively) contain additional domains which contribute to their larger size (7,8,15,24,28).…”

Structural and functional relationships in two families of β‐1,4‐glycanases

“…Two transformants were used to prepare single-stranded DNA [I 51. DNA sequencing was performed using T7 DNA polymerase with 7-deaza-dGTP instead of dGTP in the nucleotide mixes, as described previously [16]. The sequencing primer was synthesised on an Applied Biosystems 380A automated DNA synthesiser and had the following sequence: S'CCGTTCCGGGACTGCGGG3' corresponding to nucleotides 631-648 of the Sau3A-SphI fragment of pCAS10 [lo].…”