Exploring the sequence diversity in glycoside hydrolase family 13_18 reveals a novel glucosylglycerol phosphorylase
In the carbohydrate-active enzyme database, GH13_18 is a family of retaining glycoside phosphorylases that act on α-glucosides. In this work, we explored the functional diversity of this family by comparing distinctive sequence motifs in different branches of its phylogenetic tree. A glycoside phosphorylase from Marinobacter adhaerens HP15 that was predicted to have a novel function was expressed and characterised. The enzyme was found to catalyse the reversible phosphorolysis of 2-O-α-d-glucosylglycerol with retention of the anomeric configuration, a specificity that has never been described before. Homology modelling, docking and mutagenesis were performed to pinpoint particular acceptor site residues (Tyr194, Ala333, Gln336) involved in the binding of glycerol. The new enzyme specificity provides additional insights into bacterial metabolic routes, being the first report of a phosphorolytic route for glucosylglycerol in a glucosylglycerol-producing organism. Furthermore, glucosylglycerol phosphorylase might be an attractive biocatalyst for the production of the osmolyte glucosylglycerol, which is currently produced on industrial scale by exploiting a side activity of the closely related sucrose phosphorylase. Family GH13_18 has clearly proven to be more diverse than was initially assumed, and the analysis of specificity-determining sequence motifs has shown to be a straightforward and fruitful tool for enzyme discovery.
KeywordsGlucosylglycerol phosphorylase Glucosylglycerol Glycoside hydrolase family GH13 Sucrose phosphorylase
We thank Natan Van Welden for the help with lab experiments.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Bolivar JM, Luley-Goedl C, Leitner E, Sawangwan T, Nidetzky B (2017) Production of glucosyl glycerol by immobilized sucrose phosphorylase: options for enzyme fixation on a solid support and application in microscale flow format. J Biotechnol 257:131–138. https://doi.org/10.1016/j.jbiotec.2017.01.019 CrossRefPubMedGoogle Scholar
- De Winter K, Dewitte G, Dirks-Hofmeister ME, De Laet S, Pelantová H, Křen V, Desmet T (2015) Enzymatic glycosylation of phenolic antioxidants: phosphorylase-mediated synthesis and characterization. J Agric Food Chem 63:10131–10139. https://doi.org/10.1021/acs.jafc.5b04380 CrossRefPubMedGoogle Scholar
- Goedl C, Sawangwan T, Mueller M, Schwarz A, Nidetzky B (2008) A high-yielding biocatalytic process for the production of 2-O-(alpha-D-glucopyranosyl)-sn-glycerol, a natural osmolyte and useful moisturizing ingredient. Angew Chem Int Ed Engl 47:10086–10089. https://doi.org/10.1002/anie.200803562 CrossRefPubMedGoogle Scholar
- Hagemann M, Ribbeck-busch K, Kla S, Hasse D, Steinbruch R, Berg G (2008) The plant-associated bacterium Stenotrophomonas rhizophila expresses a new enzyme for the synthesis of the compatible solute glucosylglycerol. J Bacteriol 190:5898–5906. https://doi.org/10.1128/JB.00643-08 CrossRefPubMedPubMedCentralGoogle Scholar
- Jeong J, Seo D, Jung J, Park J, Baek N, Kim M, Park C (2014) Biosynthesis of glucosyl glycerol, a compatible solute, using intermolecular transglycosylation activity of amylosucrase from Methylobacillus flagellatus KT. Appl Biochem Biotechnol 173:904–917. https://doi.org/10.1007/s12010-014-0889-z CrossRefPubMedGoogle Scholar
- Krieger E, Vriend G, Spronk C (2018) YASARA—Yet Another Scientific Artificial Reality ApplicationGoogle Scholar
- Sanchis J, Fernández L, Carballeira JD, Drone J, Gumulya Y, Höbenreich H, Kahakeaw D, Kille S, Lohmer R, Peyralans JJ-P, Podtetenieff J, Prasad S, Soni P, Taglieber A, Wu S, Zilly FE, Reetz MT (2008) Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates. Appl Microbiol Biotechnol 81:387–397. https://doi.org/10.1007/s00253-008-1678-9 CrossRefPubMedGoogle Scholar
- Schrödinger LLC (2018) The PyMOL Molecular Graphics System, version 2.0Google Scholar
- Schwarz A, Goedl C, Minani A, Nidetzky B (2007) Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of α,α-trehalose. J Biotechnol 129:140–150. https://doi.org/10.1016/j.jbiotec.2006.11.022 CrossRefPubMedGoogle Scholar
- Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. https://doi.org/10.1038/msb.2011.75 CrossRefPubMedPubMedCentralGoogle Scholar
- Stam MR, Danchin EGJ, Rancurel C, Coutinho PM, Henrissat B (2006) Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of α-amylase-related proteins. Protein Eng Des Sel 19:555–562. https://doi.org/10.1093/protein/gzl044 CrossRefPubMedGoogle Scholar