Domain analysis of a modular alpha-L-Arabinofuranosidase with a unique carbohydrate binding strategy from the fiber-degrading bacterium Fibrobacter succinogenes S85.
Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique β-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher k(cat) for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the K(m) for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.
Yoshida
Shosuke
S
Energy Biosciences Institute, Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801, USA.
Hespen
Charles W
CW
Beverly
Robert L
RL
Mackie
Roderick I
RI
Cann
Isaac K O
IK
D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism / Q000502:physiology*; D045848:Fibrobacter / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013696:Temperature; D014990:Xylans / Q000737:chemistry / Q000378:metabolism
D014990:Xylans; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase