Nucleotide sequence and transcriptional analysis of the celD β-glucanase gene from Ruminococcus flavefaciens FD-1

Abstract

The nucleotide sequence of the celD gene, which encodes endoglucanase and xylanase activity, from Ruminococcus flavefaciens FD-1 was determined. The DNA sequence of celD contains an open reading frame of 1215 nucleotides that encodes a polypeptide of 405 amino acids with a molecular mass of 44,631 Da. The primary amino acid sequence of CelD was screened against the GenBank data base for similar polypeptide sequences and the analysis indicated that CelD has common features with endoglucanases from the family E cellulases. Both hydrophobic cluster and BESTFIT (Genetics Computer Group (University of Wisconsin) package) analyses confirmed this relationship. Pairwise alignments using BESTFIT revealed that CelD was most closely related to endE4 from Thermomonospora fusca over a 160 amino acid window. The histidine, aspartate, and glutamate residues identified as being essential for catalytic activity in family E cellulases are conserved in CelD. A Shine-Dalgarno-like sequence was present 5 base pairs (bp) upstream of the translation start site. Primer extension analysis indicated that different transcription initiation sites are used to initiate transcription of celD in Escherichia coli and R. flavefaciens. In the case of R. flavefaciens the transcription initiation site is at a T residue (nucleotide 273) 16 bp upstream from the translational start site. A region resembling a sigma 70-like-10 promoter sequence is present upstream from the transcription initiation site but there is no apparent-35 region. In contrast, transcription in E. coli is initiated at a C residue 258 bp upstream from the translational start site and a sequence resembling a omega 70-like-10 region is present 5 bp upstream of this residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

“…This processive endoglucanase was up-regulated 4.49 fold. CelA (ORF00507) and CelD (ORF01899) were unaffected (1.10 fold) and up-regulated (4.93 fold) respectively (Table S10), which is consistent with previous results [18], [58]. CelB (ORF01869) was unaffected (1.13 fold; Table S9), which contradicts previous data that indicated that it was inducible by cellulose [17], [58], [59].…”

“…Difficulties were encountered in initial fractionation of these enzymes as they appeared to exist in high-molecular-weight protein complexes resembling cellulosomes [12], [13], and enzymatic activity was lost rapidly when the complexes were disrupted [12]. Individual β-glucanase genes ( celA , celB , celC , and celD ) were cloned from R. flavefaciens FD-1 with a view to studying their regulation [14], [15], [16], [17], [18]. Meanwhile, parallel studies in the related R. flavefaciens strain 17 also led to the sequence analysis of a number of xylanases and cellulases.…”

EndB, a Multidomain Family 44 Cellulase from Ruminococcus flavefaciens 17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another R. flavefaciens Protein via a Dockerin Domain

“…On the other hand, there are many reports of single-domain endoglucanases from R. flavefaciens and R. albus that are smaller than 50 kDa (30,33,40,41,42). This may suggest that not all cellulases are cellulosome associated in ruminococci.…”

“…Analysis of cloned polysaccharidase genes has, however, produced somewhat conflicting evidence concerning the molecular organization of these enzymes, and the mechanisms by which they might attach to their substrate and to the cell surface have thus remained unclear. Many of the cellulase genes first isolated from R. flavefaciens and R. albus (7,18,30,33,40,41) were reported to encode single domain enzymes smaller than 50 kDa that carry no obvious substrate binding domains, and no regions that might be responsible for the types of protein-protein interactions that are found, for example, in the assembly of cellulosome complexes from Clostridium spp. (3, 4, 9).…”

Cloning, expression, purification, and characterization of a glycoside hydrolase family 44 cellulase from Clostridium acetobutylicum in Escherichia coli

“…EndB is a protein of 808 amino acids with a domain structure consisting of a signal peptide, a GH44 catalytic domain, an unknown CBM type, and a type I dockerin domain ( Figure 1) (Rincón et al 2001). CelB is a sequence of 827 amino acids for a multidomain β-glucanase protein, although this putative protein is also reported as having only 632 amino acids (Vercoe et al 1995, Warner 2007.…”