Dividing the Large Glycoside Hydrolase Family 43 into Subfamilies: a Motivation for Detailed Enzyme Characterization

Abstract

dThe rapid rise in DNA sequencing has led to an expansion in the number of glycoside hydrolase (GH) families. The GH43 family currently contains ␣-L-arabinofuranosidase, ␤-D-xylosidase, ␣-L-arabinanase, and ␤-D-galactosidase enzymes for the debranching and degradation of hemicellulose and pectin polymers. Many studies have revealed finer details about members of GH43 that necessitate the division of GH43 into subfamilies, as was done previously for the GH5 and GH13 families. The work presented here is a robust subfamily classification that assigns over 91% of all complete GH43 domains into 37 subfamilies that correlate with conserved sequence residues and results of biochemical assays and structural studies. Furthermore, cooccurrence analysis of these subfamilies and other functional modules revealed strong associations between some GH43 subfamilies and CBM6 and CBM13 domains. Cooccurrence analysis also revealed the presence of proteins containing up to three GH43 domains and belonging to different subfamilies, suggesting significant functional differences for each subfamily. Overall, the subfamily analysis suggests that the GH43 enzymes probably display a hitherto underestimated variety of subtle specificity features that are not apparent when the enzymes are assayed with simple synthetic substrates, such as pNP-glycosides. Carbohydrates serve a range of functional purposes in biological systems, including energy storage, signal transduction, and intracellular trafficking, among others (1). Importantly, carbohydrates are the main end product of plant primary production, representing a large of majority of carbon fixation by plants (2). As a photosynthetically renewable form of fixed carbon, plant biomass represents a prime target for the replacement of petroleumderived fuels for future sustainability efforts. The enzymatic degradation and modification of carbohydrates have thus been cast to the forefront of biofuel production research (3).As functional efforts to discover plant cell wall polysaccharide (PCWP)-degrading enzymes identify novel activities and mechanisms (4, 5), it is important to derive and maintain a concise classification system for these enzymes. A sequence-based classification of carbohydrate-active enzymes (CAZymes) began in 1991 (6), with the classification of 35 families of glycoside hydrolases (GHs). Today the CAZy database (7) comprises 5 separate enzyme classes, namely, the aforementioned GHs, glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and auxiliary activities (AAs), as well as associated carbohydrate binding modules (CBMs), that together correspond to over 530,000 individual sequences (at the time of submission of this article). The largest of these classes are the glycoside hydrolases, currently represented by over 241,000 sequences classified into 133 families based on amino acid sequence similarity.The rapid advancements in DNA sequencing over the past decade have exponentially increased the number of sequences assigned to each family. Henc...

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

“…Thermophilic and mesophilic enzymes abound in the large subcluster III-D; this group has the most biochemically characterized members, and within this group is CpAbf51B. To obtain information about the phylogenetic properties of CpAbn43A, the phylogeny of this GH family, reported by Mewis et al (28), was reconstructed; however, in this case, the polypeptide sequence of CpAbn43A was included in the analysis. By using this method, CpAbn43A was identified as a member of the proposed subfamily GH43_4.…”

“…Multiple-sequence alignment was performed by MUSCLE (64). To generate high-quality and relevant alignments, MAFFT (65) was used to iteratively remove highly dissimilar sequences, as described previously (28). Following these quality control measures, 1,414 sequences remained.…”

Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, β- d -Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities

“…1, the GH43 catalytic domain revealed high sequence identity to extracellular enzymes of C. stercorarium XylA (74%) (13) and P. woosongensis XylC (73%) (14), and moderate sequence identity to uncultured bacterium r_09 GH43 enzyme (47%) (7). All these enzymes belong to subfamily 35 of GH43 (20) and consist of a GH43 catalytic domain and a CBM6. The GH43 enzyme of uncultured bacterium r_09 has a 30-amino-acid stretch (amino acids 172 to 181) that was not found in the other enzymes.…”

“…According to the CAZy database, multifunctional xylanolytic enzymes are represented in GH43, including bifunctional ␤-xylosidase/␣-L-arabinofuranosidase from Clostridium stercorarium (13), Paenibacillus woosongensis (14), Humicola insolens (15), Thermoanaerobacter ethanolicus (16), Phanerochaete chrysosporium (17), and Thermotoga thermarum (18); trifunctional endo-xylanase/␤-xylosidase and ␣-Larabinofuranosidase from Paenibacillus curdlanolyticus B-6 (19); and multifunctional ␤-xylosidase/␣-L-arabinofuranosidase/␤-1,4 lactase/␣-1,6 raffinase/␣-1,6 stachyase/␤-galactosidase/␣-1,4 glucosidase from a cow rumen microbial consortium (7). GH43 is subdivided into subfamilies (1 to 37) due to the sequence diversity of its enzymes (20). Although most of the enzymes in this family characterized so far are derived from fungi and bacteria, GH43 includes enzymes from Archaea and higher plants.…”

Localizing gene regulation reveals a staggered wood decay mechanism for the brown rot fungus Postia placenta

“…Our observation of increased gene transcription and enzyme activity for α-Larabinofuranosidase behind the hyphal front offers a representative example. This α-L-arabinofuranosidase is an enzyme within a family (GH43) that contains a great deal of subtle variation, not fully captured by assays that use simple synthetic substrates (42). These GH43s are expanded in many wood-degrading fungi, including Schizophyllum commune (43).…”