Structure of the Clostridium thermocellum gene licB and the encoded β‐1,3‐1,4‐glucanase

Abstract

The nucleotide sequence of the Clostridium thermocellum gene licB, coding for a thermoactive β‐1,3‐1,4‐glucanase, has been determined. The gene is located downstream, but in opposite orientation to the β‐glucosidase gene bglA. A coding region of 1002 bp is flanked by canonical promoter and transcription terminator sequences. The primary translation product of the licB gene has a predicted molecular mass of 37896 Da. The protein sequence can be divided into several discrete segments: an N‐terminal signal peptide, a catalytic region, a segment rich in Pro and Thr residues and a C‐terminal reiterated domain. The catalytic region shows close similarity to lichenases of bacilli (52 – 58% identity) and Fibrobacter succinogenes (35% identity), but is unrelated to barley β‐1,3‐1,4‐glucanases. It consists of two domains, which in the case of the F. succinogenes lichenase are arranged in reversed order to that of C. thermocellum and Bacillus lichenases. The C‐terminal reiterated domain of C. thermocellum lichenase is homologous to the duplicated non‐catalytic domain of endo‐β‐1,4‐glucanases and xylanase Z from the same organism. This domain is considered a characteristic feature of clostridial cellulases organized as multienzyme complex (cellulosome). The β‐1,3‐1,4‐glucanase encoded by the licB gene might therefore be an additional enzyme component of the C. thermocellum cellulosome.

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313

“…GlyR1 affected the expression of licB (Clo1313_2022), containing a glycoside hydrolase 16 domain. This gene encodes a lichenase (␤-1,3-1,4-glucanase), which has been demonstrated to localize to the cellulosome (38) and cleaves ␤-1,4 linkages adjacent to ␤-1,3 linkages in mixed ␤-glucans, such as barley ␤-glucans and lichenin (39,40). LicB has no activity against ␤-1,3-␤-1,6 linkages such as laminarin (39) and so has a function distinct from that of the LicA/CelC enzymes regulated by GlyR3.…”

Cellulase, Clostridia, and Ethanol

“…These duplicated sequences, now called docking sequences or dockerins (18), are not essential for catalytic activity (113) but are responsible for the binding of the respective cellulosomal enzymes, e.g., endoglucanase D (CelD) and xylanase Z (XynZ), to one or more of the nine cohesins of CipA (186). Dockerin domains consist of two very similar segments of 22 to 24 residues connected by a peptide containing 8 to 17 amino acid residues; they are over 65% identical among the different subunits of the cellulosome (27,28,292,327). Two types of dockerin exist: type I (186), which anchors catalytic subunits to scaffoldin, and type II, which anchors scaffoldin and free enzymes to the cell surface.…”

Directed Mutagenesis of Specific Active Site Residues onFibrobacter succinogenes1,3–1,4-β-d-Glucanase Significantly Affects Catalysis and Enzyme Structural Stability

“…A F. succinogenes 1,3-1,4-␤-D-glucanase was isolated and investigated by Erfle and co-workers (15,16). This enzyme consists of a protein sequence with a circular permutation in which two highly conserved catalytic domains (namely A and B) of the enzyme are in a reverse orientation as compared with that of other 1,3-1,4-␤-D-glucanases (6,8,16). A 5 times repeated segment, PXSSSS, was only observed in the C-terminal, nonhomologous region of the amino acid sequence of the Fibrobacter enzyme relative to the bacilli or other bacterial and fungal 1,3-1,4-␤-D-glucanases.…”