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 temperature of CcGP was measured by samples were also taken after incubation for 24 h. Reactions differential scanning fluorimetry [29] in a CFX Connect96 in the phosphorolytic direction were performed with 20 cycler (Biorad) using the FRET channel. Twenty µL of mM donor, 50 mM phosphate and 0.1 mg/mL purified sample containing 1 mg/mL purified enzyme was mixed enzyme and samples were analysed with the colorimetric with 10 µL of Sypro Orange in a 1/400 dilution. The assay for Glc1P-determination. Reactions with alternative temperature was gradually increased from 20 °C to 95 °C donors (Table S3) were analysed by HPAEC. with 1 °C/min increments. Measurements were performed in triplicate. The melting temperature was determined in the CFX Manager software (Biorad) as the inflection point of the melting curve, calculated as the minimum value of 3 Results its negative first derivative. 3.1 Searching the subfamily GH13_18 for new functions 2.6 Detection of reaction products The key to differentiating the various phosphorylase The phosphomolybdate assay was used to measure the specificities in subfamily GH13_18 is a detailed comparison release of inorganic phosphate [30]. Glc1P released by of active-site sequence motifs that are fully conserved phosphorolysis could be measured using a coupled within certain clades of the phylogenetic tree, but evidently enzymatic assay based on the reduction of NAD+ in different in other clades (Fig. 1). For example, the catalytic the presence of phosphoglucomutase and glucose acid/base glutamate is always succeeded by threonine and 6-phosphate dehydrogenase [31]. Reactions were asparagine in the clade that contains the characterised also monitored by high performance anion exchange glucosylglycerate phosphorylase sequences. In all other chromatography (HPAEC; Dionex ICS-6000, Thermo known GH13 phosphorylase sequences, that glutamate is Scientific) with a CarboPac PA20 pH-stable column and consistently followed by a small hydrophobic residue and pulsed amperometric detection. A 10-µL sample was histidine. Mutagenesis studies have confirmed that these analysed at a constant flow rate of 0.5 mL/min at 30 °C. asparagine or histidine sidechains are very important The eluent composition gradually changed from 0 mM for activity of glucosylglycerate or SPs, respectively NaOH to 100 mM NaOH during 15 min. Then, the NaOAc [9,32]. Similarly, glucosylglycerol phosphorylase-like concentration was gradually increased from 0 mM to sequences can be distinguished from others by the 300 mM during the next 10 min. Finally, the column conserved VGA motif in one of the acceptor site loops was equilibrated with water for 6 min. Prior to analysis, (loop A; motif at positions 331-333 in the Marinobacter all reactions were inactivated by the low pH of the assay adhaerens enzyme). The alanine residue is predicted to mixture (phosphomolybdate assay) or by heating the participate in hydrophobic interaction with the C1’ carbon sample at 95 °C for 5 min (other detection methods). of glucosylglycerol [10]. In the neighbouring clades of sucrose and sucrose 6F-phosporylases, that alanine is always replaced by aspartate. 2.7 Screening of putative substrates Upon inspection of the subfamily multiple sequence alignment, a small group of putative phosphorylases, For screening putative acceptor substrates in the synthetic found exclusively in Corallococcus species, was direction, enzyme purified by Ni-NTA chromatography discovered to have a different set of amino acids at the (0.1 mg/mL or 1.6 µM) was incubated with 30 mM Glc1P aforementioned specificity-determining positions (Fig. 1). and a candidate substrate (list and concentrations in At the second positions following the catalytic glutamate, Table S2) in 50 mM MES buffer (pH 6.5). To ensure that these sequences have an aspartate residue instead of the A GH13 glycoside phosphorylase with unknown substrate specificity from Corallococcus coralloides 35

Figure 1: (a) Phylogenetic tree of protein sequences classified in subfamily GH13_18. A selection of representatives that have been characterised and reported in literature are indicated with their UniProt IDs. (b) Simplified phylogenetic tree showing the sequence logo of two specificity-determining loops for the proteins in the branch. The acid/base loop and loop A refer to positions 274-277 and 376-384, respectively, in the Corallococcus coralloides putative phosphorylase (UniProt ID: H8N0X1). Grey circle, Bifidobacterium-like SPs; white circle, lactic acid bacteria-like SPs; black circle, glucosylglycerate phosphorylases; star, glucosylglycerol phosphorylases; white triangle, TtSPP-like sucrose 6F-phosphate phosphorylases; grey diamond, Corallococcus putative GPs. asparagine present in glucosylglycerate phosphorylases, sequence showed no predicted N-terminal signal peptide or the histidine present in others. Further, only the according to the SignalP-5.0 server [27], and no predicted Corallococcus sequences possess a GEXRPYE motif in the disulphide bonding cysteines according to the DISULFIND acceptor loop A. These unusual sequence patterns were server [28]. CcGP, provided with a C-terminal His6-tag, was presumed to be indicative of functional divergence from recombinantly expressed in E. coli and purified by affinity other enzymes of the GH13_18 subfamily. Therefore, it was chromatography. A yield of 12 mg soluble enzyme was deemed worthwhile to express a gene from this atypical obtained from lysates of a 250-mL cell culture, which is group of enzymes to evaluate their substrate preference. quite high compared to the expression levels of known GH13_18 phosphorylases. The thermodynamic stability of CcGP was checked

3.2 Expression and characterisation by measuring its melting temperature (TM) by differential scanning fluorimetry (Fig. S1). The melting temperature All sequences in the clade of interest of the subfamily correlates with the tendency of a protein to reversibly

GH13_18 phylogenetic tree originate from Corallococcus unfold as a function of temperature [34]. With a TM of 44 species, feature the same sequence motifs in subsite +1 °C, CcGP can be considered a mesophilic enzyme. and have a mutual sequence identity that exceeds 85% The activity of CcGP was first examined on natural (Table S4). Hence, they most likely exhibit the same substrates of GH13_18 phosphorylases in the synthetic substrate specificity. A gene from the validly described direction, using Glc1P as glycosyl donor and fructose, type strain C. coralloides DSM 2259, of which the complete fructose 6-phosphate, glycerol or glyceric acid as 10.08 Mbp genome is publically available [33], was putative glycosyl acceptor. No transglucosylation activity chosen to represent the clade. This putative GP from could be observed for any of the compounds, except for C. coralloides (CcGP; UniProt ID: H8N0X1) shares little fructose 6-phosphate, where minor amounts of sucrose sequence identity with other characterised enzymes from 6F-phosphate were synthesised upon prolonged incubation the subfamily, such as the Bifidobacterium adolescentis (< 0.05 U/mg) (Fig. S2). Reactions in the phosphorolytic SP (20%), the Marinobacter adhaerens glucosylglycerol direction confirmed these results. Correspondingly, CcGP phosphorylase (21%) or the Meiothermus silvanus is not a sucrose, sucrose 6F-phosphate, glucosylglycerol or glucosylglycerate phosphorylase (30%). Moreover, the glucosylglycerate phosphorylase, thereby setting it apart 36 Jorick Franceus, Tom Desmet

Figure 2: (a) Subsite –1 and (b) subsite +1 of Bifidobacterium adolescentis SP (BaSP; PDB code: 2GDU). Residues interacting with phosphate, the glucosyl moiety or the fructosyl moiety of sucrose are shown in yellow, green and cyan, respectively. The catalytic residues are shown in purple. (c) Partial sequence alignment of GH13_18 phosphorylases. Purple dots below sequences indicate catalytic residues. Green, cyan and yellow dots indicate positions involved in recognition of the glucosyl, fructosyl or phosphate group in BaSP. Conserved residues are shown in bold. A position is framed blue if the majority of its residues are similar. Those similar residues are shown in red. Residue numbers are shown for BaSP and Corallococcus coralloides GP (CcGP). Secondary structures are indicated above the sequences with squiggles (α-helices), arrows (β-sheets) or a T character (turns). LmSP, Leuconostoc mesenteroides SP (Uniprot ID: Q59495); TtSPP, Thermoanaerobacter thermosaccharolyticum sucrose 6F-phosphate phosphorylase (D9TT09); MaGGoP, M. adhaerens glucosylglycerol phosphorylase (E4PMA5); MsGGaP, Meiothermus silvanus glucosylglycerate phosphorylase (D7BAR0); CcGP, Corallococcus coralloides GP (H8N0X1). from all other GH13_18 phosphorylases that have been moiety or phosphate are well documented due to the characterised so far. In an attempt to ascribe a function availability of crystal structures of the B. adolescentis SP to CcGP, an extensive acceptor substrate screening was in various stages of its catalytic cycle [35,36], accompanied performed in the synthetic direction. More than seventy by mutagenesis studies that have clarified the function of different candidate substrates were tested, including many active site residues [32,37-39]. Based on a multiple common monosaccharides and some of their isomers, α- sequence alignment of several GH13_18 sequences, it and β-bonded disaccharides, oligo- and polysaccharides, can be concluded that the positions that are associated phosphorylated sugars, polyols, acids, glucosides with glucosyl binding in subsite –1 are occupied by the and phenolic or polyphenolic compounds (Table S2). same residues in CcGP and in the other phosphorylases Unfortunately, no significant activity could be detected (Fig. 2). The phosphate-binding residues show slightly towards any of these. Additionally, no hydrolytic reaction more variation across the different sequences, but the could be observed during incubation with only Glc1P. sidechains present at these positions in CcGP are fully in The lack of clear activity raised the question of line with expectations. Based on this comparison, it seems whether Glc1P is the correct donor substrate of CcGP, that the clear-cut differences in the acceptor site do not prompting a closer look at residues in subsite –1. The extend to the donor site, and therefore there is no reason residues that engage in interactions with the glucosyl to assume that the donor substrate preference of CcGP A GH13 glycoside phosphorylase with unknown substrate specificity from Corallococcus coralloides 37 would not match that of the other enzymes. Regardless, Considering the high metabolic diversity associated with a few alternative compounds were tested as donor, the synthesis of these metabolites and the degradation of including a few nucleotide-activated sugars to check prey macromolecules, it should come to no surprise that for glycosyltransferase activity, but no activity could be Corallococcus has a large accessory pan-genome, probably detected (Table S3). carrying a pool of genes with novel functions [44]. Bacteriolytic myxobacteria can utilize several polysaccharides and maltotriose as carbon source, but 4 Discussion mono- or disaccharides do not stimulate growth [45]. Several new glycoside hydrolases from Corallococcus In this study, we identified a putative GP from Corallococcus species, including some from family GH13, have already coralloides DSM 2259 that does not have the typical been characterised in the past few years [46-51]. Those characteristics of the previously studied phosphorylases GH13 hydrolases indeed process maltooligosaccharides, in subfamily GH13_18 of the CAZy database. CcGP and but the GH13 phosphorylase discussed in this work did not its close homologs show a unique pattern of sequence appear to show such activity. CcGP may thus be involved in conservation in subsite +1, whereas the residues present the processing of more complex carbohydrates, or of one at substrate-binding positions in subsite –1 are identical of the organism’s many secondary metabolites. It would to those in the other phosphorylases. CcGP does not be interesting to monitor the expression level of CcGP display any sucrose, glucosylglycerol or glucosylglycerate under different growth conditions in vivo, as that may phosphorylase activity, and minor sucrose 6F-phosphate provide clues on its function. For instance, it had been phosphorylase activity was only detected after prolonged suggested that a putative SP from Gimesia maris could be incubation. The latter activity is not large enough to be involved in the synthesis of the osmolyte glucosylglycerate relevant in a physiological context, but does prove that based on the transcript abundance of its gene under the protein is catalytically active and capable of binding specific stressful conditions, and it later turned out that Glc1P, as was also inferred from the strict conservation this putative SP is in fact probably a glucosylglycerate of Glc1P-binding residues. Unfortunately, however, an phosphorylase [9,52]. extensive candidate substrate screening failed to bring Although the real substrate of CcGP has not yet been its true natural function to light. In any case, the absence identified, the present study underlines the wealth of of activity towards the large and diverse range of tested natural variety in several areas. First, the finding of CcGP compounds implies that the enzyme is nowhere near yet again expands the collection of functionally divergent as promiscuous as the related SPs, and presumably enzymes in GH13_18. The idea that the subfamily consists has a rather strict specificity like glucosylglycerol and only of SPs, which was a reasonable assumption merely glucosylglycerate phosphorylases do. five years ago, no longer holds true today. The recent Homologs of the enigmatic enzyme are found accumulation of new information about the variations exclusively in other Corallococcus species. These in sequence and activity in these carbohydrate-active species belong to a fascinating group of predatory enzymes may prove to be valuable in future engineering microorganisms called myxobacteria [40,41]. They projects. In a broader context, CcGP and the related live an intriguingly complex social lifestyle, forming enzymes that were recently characterised also expand the multicellular groups, commonly referred to as swarms, larger oligo-1,6-glucosidase subfamily that GH13_18 is part that move together by gliding over solid surfaces. of, together with subfamilies 4, 16, 17, 23, 29, 30, 31, 34 and These swarms cooperatively hunt and feed on prey by 35 [53,54]. The sequence of CcGP contains the characteristic secreting lytic enzymes and antibiotics, and subsequently QpDln motif of the oligo-1,6-glucosidase subfamily, as well consuming the released nutrients. When experiencing as the distinguishing sequence features at other conserved starvation, three-dimensional macromolecular fruiting regions that are only found in the subfamily’s transferases bodies are formed, where some cells are converted (Fig. S3). Finally, the fact that homologs of CcGP have only into spores, while others are altruistically sacrificed to been encountered in Corallococcus species is another provide food for the remaining survivors. A plethora of indication that it is worth scanning the genome of these secondary metabolites are produced by myxobacteria to myxobacteria for enzymes with new functions, even when support their unconventional life cycle, and as a result, at first glance, the enzymes resemble ones that have the organisms are widely regarded as a prolific source already been characterised before. of natural products, many of which exhibit captivating biological activities with unusual modes-of-action [42,43]. 38 Jorick Franceus, Tom Desmet

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