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Download Article (PDF) Amylase 2019; 3: 32–40 Research Article Jorick Franceus, Tom Desmet* A GH13 glycoside phosphorylase with unknown substrate specificity from Corallococcus coralloides https://doi.org/10.1515/amylase-2019-0003 received June 27, 2019; accepted August 6, 2019. subfamily 18; Glc1P, α-d-glucose 1-phosphate; GP, glycoside phosphorylase; HPAEC, high performance anion exchange Abstract: Glycoside phosphorylases in subfamily GH13_18 chromatography; MES, morpholinoethanesulfonic acid; of the carbohydrate-active enzyme database CAZy catalyse SP, sucrose phosphorylase. the reversible phosphorolysis of α-glycosidic bonds. They contribute to a more energy-efficient metabolism in vivo, and can be applied for the synthesis of valuable glucosides, 1 Introduction sugars or sugar phosphates in vitro. Continuing our efforts to uncover new phosphorylase specificities, we identified Recent years have witnessed an increased understanding an enzyme from the myxobacterium Corallococcus of the functional diversity of glycoside phosphorylases coralloides DSM 2259 that does not feature the (GPs) in family GH13 of the carbohydrate-active enzyme signature sequence patterns of previously characterised database CAZy (http://www.cazy.org/) [1], generally phosphorylases. The enzyme was recombinantly known as the α-amylase family. These enzymes catalyse expressed and subjected to substrate screening. Although the reversible phosphorolysis of glycosidic bonds to form it was confirmed that the Corallococcus phosphorylase sugar 1-phosphates [2-4], and do so with retention of the does not have the same substrate specificity as other anomeric configuration due to their double displacement phoshorylases from subfamily GH13_18, its true natural mechanism [5]. There are 42 subfamilies in GH13 at substrate remains a mystery for now. Myxobacteria have present, but only numbers 3 and 18 harbour characterised been widely investigated as producers of numerous GPs. When subfamily 18 (GH13_18) was first defined, the bioactive secondary metabolites for decades, but little only characterised enzymes it contained were sucrose research has been conducted on myxobacterial proteins. phosphorylases (SP; EC 2.4.1.7), of which the first report The present study exemplifies the untapped metabolic dates back to the 1940s [6,7]. These enzymes catalyse the activities and functional diversity that these fascinating reversible phosphorolysis of sucrose, forming α-d-glucose organisms may have left to show. 1-phosphate (Glc1P) and fructose. No new specificities from evolutionarily closely related sequences were Keywords: glycoside phosphorylase; Corallococcus found until 2014, when Verhaeghe et al. [8] described coralloides; glycoside hydrolase family GH13; sucrose a phylogenetic neighbour that was specialised in the phosphorylase; myxobacteria. phosphorolysis of sucrose 6F-phosphate (EC 2.4.1.329), albeit with a very broad substrate specificity. Rational searches for other new functions led to the recent discovery Abbreviations of glucosylglycerate phosphorylases (EC 2.4.1.352) [9], glucosylglycerol phosphorylases (EC 2.4.1.359) [10] CcGP, glycoside phosphorylase from Corallococcus and sucrose 6F-phosphorylases with a strict substrate coralloides; GH13_18, glycoside hydrolase family 13 specificity [11,12]. Much of the interest in GH13 phosphorylases is driven by their outstanding potential for synthesising α-glycosidic *Corresponding author: Tom Desmet, Centre for Synthetic Biology, bonds. The Glc1P reaction product has a high energy Department of Biotechnology, Ghent University, Coupure Links 653, content, allowing the degradative reaction to be reversed B-9000 Ghent, Belgium, E-mail: [email protected] easily and efficiently. SPs have two additional advantages Jorick Franceus, Centre for Synthetic Biology, Department of in that regard. First, they can catalyse transglucosylation Biotechnology, Ghent University, Coupure Links 653, B-9000 Ghent, reactions starting from sucrose, which is equally reactive Belgium Open Access. © 2019 Jorick Franceus, Tom Desmet, published by De Gruyter. This work is licensed under the Creative Commons Attribution alone 4.0 License. A GH13 glycoside phosphorylase with unknown substrate specificity from Corallococcus coralloides 33 as Glc1P, but much cheaper. Second, SPs typically show a 2.2 Sequence analysis high degree of acceptor promiscuity and therefore, they can transfer the donor glucosyl group to a large variety All full-length sequences from subfamily 18 of family GH13 of small molecules, such as polyols [13], sugar acids [14], were extracted from the CAZy database [1] and subsequently monosaccharides [15-18] or phenolic antioxidants [19]. aligned using ClustalO with default parameters [22]. A Nevertheless, the other phosphorylases from the family maximum-likelihood phylogenetic tree was constructed can be of interest as well, because they can be coupled with PhyML using default parameters [23] and visualised to SP for straightforward generation of the required Glc1P in iTOL v4 [24]. Homologous sequences were found using intermediate in situ [20,21]. the basic local alignment search BLAST tool (https:// The recently unearthed phosphorylase specificities blast.ncbi.nlm.nih.gov/). Sequence logos were generated also cast new light on a few catabolic routes. The genes using WebLogo 3 [25]. Multiple sequence alignments encoding sucrose 6F-phosphate phosphorylases are were visualised using ESPript [26]. The absence of signal located in an operon together with a phosphofructokinase peptides and disulphide bonding cysteines was predicted and genes that encode a putative phosphoenolpyruvate- by SignalP-5.0 [27] and DISULFIND [28], respectively. dependent transport system. Their concerted action implies a new pathway for catabolism of sucrose, where the sugar is phosphorylated during uptake and 2.3 Gene cloning and transformation phosphorolysed into Glc1P and fructose 6-phosphate; the latter subsequently being transformed into fructose The sequence for the Corallococcus coralloides DSM 2259 1,6-diphosphate by the kinase [8,11]. With regard to the GPe (CcGP; UniProt ID: H8N0X1) was codon optimised glucosylglycerate and glucosylglycerol phosphorylases, for E. coli (sequence in Table S1), synthesised and they were the first known enzymes capable of breaking subcloned into a pET21a expression vector at NheI and down these compatible solutes in numerous bacterial XhoI restriction sites by Life Technologies (Belgium). The phyla, and solved the question of where the metabolic obtained plasmid was transformed in E. coli BL21(DE3) sinks of these compounds can be found [9]. electrocompetent cells. The aforementioned examples emphasise how the search for new kinds of phosphorylases can create opportunities in industrial biotechnology and expose 2.4 Protein expression and purification missing pieces of metabolic puzzles. In a continued effort to uncover the full diversity of enzyme functionalities CcGP was expressed by inoculating 2% of an overnight in the subfamily GH13_18, the database was scanned culture in a 1-L shake flask containing 250 LB medium with for more sequences with alternative amino acids at 100 µg/mL ampicillin and incubating the culture at 37 °C specificity-determining positions, in comparison to the and 200 rpm until the OD600 reached ~0.6. Expression was familiar sucrose, sucrose 6F-phosphate, glucosylglycerol induced by adding isopropyl β-d-1-thio-galactopyranoside or glucosylglycerate phosphorylases. This study describes to a final concentration of 0.1 mM and continuing an enzyme from the myxobacterium Corallococcus incubation at 18 °C for 16 h. The culture was centrifuged coralloides with an unusual set of residues in the active and the pellet was frozen at –20 °C for at least 2 hours. site. Cell pellets were lysed by adding 8 mL lysis buffer containing 0.1 mM phenylmethylsulfonyl fluoride, 1 mg/mL lysozyme, 10 mM imidazole and 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5), 2 Materials and methods and incubating the mixture at 4 °C for 30 min. The extract was sonicated three-times for 3 min (Branson Sonifier 2.1 Materials 250, level 3, 50% duty cycle) and centrifuged to remove cell debris. The cell-free extracts were further purified All chemicals were obtained from Sigma-Aldrich, Merck by Ni-NTA affinity chromatography as described by the or Carbosynth unless noted otherwise and were of the supplier (HisPur resin, Thermo Scientific). Afterwards, the highest purity. Glc1P was produced in-house using the SP buffer was exchanged to 50 mM MES (pH 6.5) using 30-kDa from Bifidobacterium adolescentis [20]. The Escherichia Amicon Ultra centrifugal filters. A NanoDrop ND-1000 coli BL21(DE3) strain was obtained from New England was used to measure protein concentration, with the Biolabs. extinction coefficient calculated by the ProtParam tool at 34 Jorick Franceus, Tom Desmet the ExPASy server (60 390 M-1cm-1; http://web.expasy.org/ even minor activities could be detected, the applied protparam/). Sodium dodecyl sulfate-polyacrylamide gel enzyme concentration was an order of magnitude higher electrophoresis (10%) was performed to verify molecular than typically required for measuring the initial activity weight and purity. of phosphorylases. Samples of 50 µL were taken every 2 min for 10 min and analysed by the phosphomolybdate assay to measure the released phosphate. Additionally, a 2.5 Differential scanning fluorimetry sample was taken after 30 min and analysed by HPAEC. For fructose, fructose 6-phosphate, glycerol and glycerate, The melting
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