Structural basis for the exocellulase activity of the cellobiohydrolase CbhA from Clostridium thermocellum.
Numerous bacterial and fungal organisms have evolved elaborate sets of modular glycoside hydrolases and similar enzymes aimed at the degradation of polymeric carbohydrates. Presently, on the basis of sequence similarity catalytic modules of these enzymes have been classified into 90 families. Representatives of a particular family display similar fold and catalytic mechanisms. However, within families distinctions occur with regard to enzymatic properties and type of activity against carbohydrate chains. Cellobiohydrolase CbhA from Clostridium thermocellum is a large seven-modular enzyme with a catalytic module belonging to family 9. In contrast to other representatives of that family possessing only endo- and, in few cases, endo/exo-cellulase activities, CbhA is exclusively an exocellulase. The crystal structures of the combination of the immunoglobulin-like module and the catalytic module of CbhA (Ig-GH9_CbhA) and that of an inactive mutant Ig-GH9_CbhA(E795Q) in complex with cellotetraose (CTT) are reported here. The detailed analysis of these structures reveals that, while key catalytic residues and overall fold are conserved in this enzyme and those of other family 9 glycoside hydrolases, the active site of GH9_CbhA is blocked off after the -2 subsite. This feature which is created by an extension and altered conformation of a single loop region explains the inability of the active site of CbhA to accommodate a long cellulose chain and to cut it internally. This altered loop region is responsible for the exocellulolytic activity of the enzyme.
Schubot
Florian D
FD
Department of Biochemistry & Molecular Biology, The University of Georgia, Athens, Georgia 30602, USA.
Kataeva
Irina A
IA
Chang
Jessie
J
Shah
Ashit K
AK
Ljungdahl
Lars G
LG
Rose
John P
JP
Wang
Bi-Cheng
BC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D044602:Cellulases / Q000737:chemistry* / Q000235:genetics; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D007136:Immunoglobulins / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013780:Tetroses / Q000737:chemistry
D007136:Immunoglobulins; D021122:Protein Subunits; D013780:Tetroses; C048468:cellotetraose; D002482:Cellulose; D044602:Cellulases; D043366:Cellulose 1,4-beta-Cellobiosidase