Biochemical and domain analyses of FSUAxe6B, a modular acetyl xylan esterase, identify a unique carbohydrate binding module in Fibrobacter succinogenes S85.
Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of beta-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser(44), His(273), Glu(194), and Asp(270), with both Glu(194) and Asp(270) functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.
Yoshida
Shosuke
S
Energy Biosciences Institute, University of Illinois, Urbana, IL 61801, USA.
Mackie
Roderick I
RI
Cann
Isaac K O
IK
D000115:Acetylesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D002942:Circular Dichroism; D019295:Computational Biology; D045848:Fibrobacter / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D014990:Xylans / Q000378:metabolism
D001426:Bacterial Proteins; D014990:Xylans; D000115:Acetylesterase; C081851:acetylxylan esterase