Characterization of two noncellulosomal subunits, ArfA and BgaA, from Clostridium cellulovorans that cooperate with the cellulosome in plant cell wall degradation.
Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases. To characterize the alpha-L-arabinosidases that are involved in hemicellulose degradation, we screened the C. cellulovorans genomic library for clones with alpha-L-arabinofuranosidase or alpha-L-arabinopyranosidase activity, and two clones utilizing different substrates were isolated. The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci. The amino acid sequences for ArfA and BgaA were related to alpha-L-arabinofuranosidase and beta-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively. Recombinant ArfA (rArfA) had high activity for p-nitrophenyl alpha-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl alpha-L-arabinopyranoside. On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl alpha-L-arabinopyranoside but also p-nitrophenyl beta-D-galactopyranoside. However, when the affinities of rBgaA for p-nitrophenyl alpha-L-arabinopyranoside and p-nitrophenyl beta-D-galactopyranoside were compared, the K(m) values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for alpha-L-arabinopyranose residues than for beta-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 beta-galactosidase. Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction. When rArfA and rBgaA were incubated with beta-1,4-xylanase A (XynA), a cellulosomal enzyme from C. cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone. These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits.
Kosugi
Akihiko
A
Section of Molecular and Cellular Biology, University of California Davis, 95616, USA.
Murashima
Koichiro
K
Doi
Roy H
RH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D002473:Cell Wall / Q000378:metabolism*; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D010944:Plants / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; C007916:hemicellulose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase