Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA

Abstract

The nucleotide sequence of the Clostridium thermocellum F7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like beta-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.

FIG. 1

Nucleotide and deduced amino acid sequences of the cbhA gene. The potential ribosome-binding site (SD) is in boldface type and underlined. A palindrome is indicated by arrows facing each other. The putative leader sequence is indicated by italic type. The segments encoding the different regions of CbhA are indicated by boxes of different patterns: ▨, CBD family IV; , Ig-like domain; ▪, catalytic domain; ▤, Fn3-like domain; ▧, CBD family III; ▥, dockerin domain. The underlined amino acids were determined for cellulosomal protein S3 by liquid-phase sequencing.

FIG. 1

Nucleotide and deduced amino acid sequences of the cbhA gene. The potential ribosome-binding site (SD) is in boldface type and underlined. A palindrome is indicated by arrows facing each other. The putative leader sequence is indicated by italic type. The segments encoding the different regions of CbhA are indicated by boxes of different patterns: ▨, CBD family IV; , Ig-like domain; ▪, catalytic domain; ▤, Fn3-like domain; ▧, CBD family III; ▥, dockerin domain. The underlined amino acids were determined for cellulosomal protein S3 by liquid-phase sequencing.

FIG. 2

Alignment of amino acid sequences of family IV CBDs of bacterial cellulases and endo-1,3-β-glucanases. Abbreviations and accession numbers: Cth-LicA, C. thermocellum LicA, X89732; Tne-LamA, Thermotoga neapolitana LamA (54), Z47974; Cth-CbhA, C. thermocellum CbhA; Cce-CelE, Clostridium cellulolyticum CelE (2), Q46002; Cfi-CenC, Cellulomonas fimi CenC (9), P14090; Sre-Cel1, S. reticuli Cel1 (42), Q05156; Tfu-E1, Thermomonospora fusca E1 (26), Q08166. Shaded boxes highlight positions where residues are conserved in five or more family members, including those of CbhA. The conserved aromatic residues are indicated by asterisks. All sequences are numbered from Met-1.

FIG. 3

Alignment of amino acid sequences of selected CBDs from family III. Abbreviations and accession numbers: Cth-CbhA, C. thermocellum CbhA; Cth-CipA, C. thermocellum CipA (12), X67506; Csa-CelB, Caldicellulosiruptor saccharolyticus CelB (41), X13602; Cst-CelZ, Clostridium stercorarium CelZ (20), X55299; Cth-CelI, C. thermocellum CelI (17), L04735; Bla-CelA, Bacillus lautus CelA (15), M76588; Eca-CelV, Erwinia carotovora CelV (33), X79241. Shaded boxes highlight positions where residues are conserved in four or more family members, including those of CbhA. The conserved aromatic residues and the residues that are implicated in Ca²⁺ binding are indicated by asterisks and solid triangles, respectively. All sequences are numbered from Met-1.

FIG. 4

HCA plots of CbhA amino acid positions 825 to 912 (A) and 914 to 1000 (B). Hydrophobic amino acids are shown as gray circles with conserved positions highlighted in dark gray. Proline residues are shown as black circles, and other helix-breaking amino acids (D, G, S, and N) found predominantly in loop regions are shown as white circles.

FIG. 5

Secondary-structure prediction of Fn3-like modules. Abbreviations and accession numbers: Tfu-E4, Thermomonospora fusca exoglucanase E4 (26), L20093; Hum-Fib, human fibronectin (34), P02751. Other abbreviations are as described in the legend to Fig. 2. Secondary-structure states of amino acids were predicted by the PREDATOR program (11) and are represented by an “E” (extended or sheet) and a dash (coil). The seven antiparallel β-strands of the 10th Fn3 module of human fibronectin are designated by the letters A to F (34) at the bottom of the figure. All sequences are numbered from Met-1.

FIG. 6

Detection of CbhA in the cellulosome of C. thermocellum. (Left panel) Western blot analysis of cellulosomal proteins detected with a polyclonal antibody raised against truncated CbhA; (right panel) SDS-PAGE of cellulosomal proteins stained with Coomassie brilliant blue. Cellulosomal subunits S1 to S14 are indicated with corresponding molecular masses.

FIG. 7

Comparison of the domain structure of cellulases of subfamily E1. Domains and regions showing significant similarity are indicated by the same pattern. Abbreviations and accession numbers: Cth-CelJ, C. thermocellum CelJ (1), D83704; Cth-CelD, C. thermocellum CelD (24), X04584; Pfl-EglA, P. fluorescens EglA (14), X12570; Fsu-EgB, Fibrobacter succinogenes EgB (6), L14436; Bfi-CelD, Butyrivibrio fibrisolvens CelD (5), X55732; aa, amino acids. Other abbreviations for the enzymes are described in the legend to Fig. 2.

FIG. 8

Average linkage cluster analysis. Similar amino acids were grouped by the classification of Risler et al. (40). The dendrogram was derived from pairwise similarity scores in accordance with the UPGMA (unweighted pair group maximum averages) method (45). Abbreviations for enzymes are described in the legends to Fig. 2 and 7.