Journal:MicroPubl Biol:000868

Gossypium hirsutum gene of unknown function Gohir.A02G131900 encodes a potential plant-specific, dual-domain exo-1,3-β-glucosidase

Gillian Hernandez, Amanda M Hulse-Kemp, and Amanda R Storm ^[1]

Molecular Tour
There are many genes of unknown function in the genomes of all organisms. By studying the predicted structure of the encoded proteins, we can better understand each protein's role and importance for life. A protein of unknown function in a number of important agricultural crops including upland cotton, referred to here as GhGH5BG-A0A1U8NW40 (Gossypium hirsutum Glycosyl Hydrolase 5 β-Glucosidase, UniProt A0A1U8NW40), has a highly confident predicted AlphaFold structure

One domain in GhGH5BG-A0A1U8NW40 is recognized by InterPro as a ‘glycoside hydrolase (Cellulase A) family 5’ domain (IPR001547). Proteins with a GH5 domain have a structure with an , which is a structure of eight alpha helices and eight parallel beta strands commonly found in enzymes, protein catalysts (Silverman et al. 2001^[2]). GH5 domain is colored in orange with alpha-beta (β/α)8 barrel in cyan and fascin-like subdomain is in red. The GH5 family contains enzymes with several known activities that hydrolyze or cut carbohydrate (glucan) sugar chains in different ways. In plants, many of these types of enzymes are involved in remodeling the plant cell wall as plants grow and develop as well as respond to stresses. One major difference between enzymes in the GH5 family is whether they cut sugars from the end of the chain (exo activity) or somewhere within the chain (endo activity). The GH5 family contains both exo and endo glucosidases but the structure of the active site can help indicate which category GhGH5BG-A0A1U8NW40 belongs to. Because endo enzymes cut in the middle of the carbohydrate chain, their active sites are shaped like an open groove or cleft to allow it to fit around the chain (such as in this endo enzyme ; PDB 1cen). However, exo enzymes cut sugars off the end of the carbohydrate chain so their active sites are shaped more like a deep pocket (such as this , PDB 3n9k). The proposed function of .

We can also support whether the GhGH5BG-A0A1U8NW40 protein is a functional exo (1,3-β-glucosidase) by from Candida albicans (PDB 3n9k). GhGH5BG-A0A1U8NW40 GH5 domain is in cyan; actin-crosslinking domain: red; homologous exo-1,3- β-glucosidase XOG1 (PDB ID 3n9k): bisque ribbon. . The 3n9k structure contains a substrate analog, . Ball-and-stick representation of substrate analog laminaritriose (element coloring) and selected side chains (GH5-invariant residues: blue; catalytic residues: purple. Residues that are found in all active GH5 enzymes, including two catalytic glutamate residues, are all conserved in GhGH5BG-A0A1U8NW40 and the placement of these residues in the model align with the 3n9k structure (Patrick et al. 2010^[3]). Such information supports the prediction that GhGH5BG-A0A1U8NW40 is a catalytically active GH5 enzyme.

In addition to the GH5 domain, the GhGH5BG-A0A1U8NW40 protein has a second domain with structural homology to a certain fold found in Fascin proteins (IPR010431). Fascins are a family of actin-crosslinking proteins found across invertebrate and vertebrate eukaryotes, including humans, that are involved in the organization of the actin cytoskeleton and cell motility. The (PDB 1dfc; each β-trefoil fold highlighted in a different color). The GhGH5BG-A0A1U8NW40 protein has one of these β-trefoil fold subdomains which is interesting because no homologs of fascin proteins are found in plants. However, other studies have found that a plant-specific subfamily of GH5 proteins all have this fascin-like domain (Opassiri et al. 2007^[4]). The GhGH5BG-A0A1U8NW40 fascin-like domain does not contain the key residues required in Fascin proteins for crosslinking actin so this unique plant domain has likely evolved a different function, perhaps a new glucan-binding ability, but experimental studies are needed to explore this interesting feature.

References

1. ↑ doi: https://dx.doi.org/10.17912/micropub.biology.000868
2. ↑ Silverman JA, Balakrishnan R, Harbury PB. Reverse engineering the (beta/alpha )8 barrel fold. Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3092-7. PMID:11248037 doi:10.1073/pnas.041613598
3. ↑ Patrick WM, Nakatani Y, Cutfield SM, Sharpe ML, Ramsay RJ, Cutfield JF. Carbohydrate binding sites in Candida albicans exo-beta-1,3-glucanase and the role of the Phe-Phe 'clamp' at the active site entrance. FEBS J. 2010 Sep 10. doi: 10.1111/j.1742-4658.2010.07869.x. PMID:20875088 doi:10.1111/j.1742-4658.2010.07869.x
4. ↑ Opassiri R, Pomthong B, Akiyama T, Nakphaichit M, Onkoksoong T, Ketudat Cairns M, Ketudat Cairns JR. A stress-induced rice (Oryza sativa L.) beta-glucosidase represents a new subfamily of glycosyl hydrolase family 5 containing a fascin-like domain. Biochem J. 2007 Dec 1;408(2):241-9. PMID:17705786 doi:10.1042/BJ20070734