An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2.
The cellulosome produced by Piromyces sp. strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water. Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90 kDa. Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested. Incubation with 0.5% (w/v) SDS or 8 M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170 kDa components. To investigate the major 90 kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing. Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi. The 2800 bp cDNA clone contained an open reading frame of 2334 bp encoding a 757-residue extracellular protein. Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family. Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase. The catalytic domain is succeeded by a putative beta-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains. Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges. The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.
Steenbakkers
Peter J M
PJ
Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands.
Ubhayasekera
Wimal
W
Goossen
Harry J A M
HJ
van Lierop
Erik M H M
EM
van der Drift
Chris
C
Vogels
Godfried D
GD
Mowbray
Sherry L
SL
Op den Camp
Huub J M
HJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D020134:Catalytic Domain; D002480:Cellulase / Q000235:genetics* / Q000302:isolation & purification*; D002874:Chromosome Mapping; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification*; D007438:Introns; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D020089:Piromyces / Q000201:enzymology* / Q000235:genetics*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D018076:DNA, Complementary; D004271:DNA, Fungal; D006026:Glycoside Hydrolases; D002480:Cellulase; C451300:cel9A protein, Pseudomonas