Identification of Euglena gracilis β-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149.
ABSTRACT: Glycoside phosphorylases (EC 2.4.x.x) carry out the reversible phosphorolysis of glucan polymers, producing the corresponding sugar 1-phosphate and a shortened glycan chain. β-1,3-Glucan phosphorylase activities have been reported in the photosynthetic euglenozoan Euglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology of E. gracilis, we identified a candidate phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 in Escherichia coli and characterized it in vitro as a β-1,3-glucan phosphorylase. BLASTP identified several hundred EgP1 orthologs, most of which were from Gram-negative bacteria and had 37-91% sequence identity to EgP1. We heterologously expressed a bacterial metagenomic sequence, Pro_7066 in E. coli and confirmed it as a β-1,3-glucan phosphorylase, albeit with kinetics parameters distinct from those of EgP1. EgP1, Pro_7066, and their orthologs are classified as a new glycoside hydrolase (GH) family, designated GH149. Comparisons between GH94, EgP1, and Pro_7066 sequences revealed conservation of key amino acids required for the phosphorylase activity, suggesting a phosphorylase mechanism that is conserved between GH94 and GH149. We found bacterial GH149 genes in gene clusters containing sugar transporter and several other GH family genes, suggesting that bacterial GH149 proteins have roles in the degradation of complex carbohydrates. The Bacteroidetes GH149 genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial β-1,3-glucan phosphorylase and uncovered a new family of phosphorylases that we name GH149.
Project description:Glycoside phosphorylases (GPs) catalyze the phosphorolysis of glycans into the corresponding sugar 1-phosphates and shortened glycan chains. Given the diversity of natural ?-(1?3)-glucans and their wide range of biotechnological applications, the identification of enzymatic tools that can act on ?-(1?3)-glucooligosaccharides is an attractive area of research. GP activities acting on ?-(1?3)-glucooligosaccharides have been described in bacteria, the photosynthetic excavate Euglena gracilis, and the heterokont Ochromonas spp. Previously, we characterized ?-(1?3)-glucan GPs from bacteria and E. gracilis, leading to their classification in glycoside hydrolase family GH149. Here, we characterized GPs from Gram-positive bacteria and heterokont algae acting on ?-(1?3)-glucooligosaccharides. We identified a phosphorylase sequence from Ochromonas spp. (OcP1) together with its orthologs from other species, leading us to propose the establishment of a new GH family, designated GH161. To establish the activity of GH161 members, we recombinantly expressed a bacterial GH161 gene sequence (PapP) from the Gram-positive bacterium Paenibacillus polymyxa ATCC 842 in Escherichia coli We found that PapP acts on ?-(1?3)-glucooligosaccharide acceptors with a degree of polymerization (DP) ? 2. This activity was distinct from that of characterized GH149 ?-(1?3)-glucan phosphorylases, which operate on acceptors with DP ? 1. We also found that bacterial GH161 genes co-localize with genes encoding ?-glucosidases and ATP-binding cassette transporters, highlighting a probable involvement of GH161 enzymes in carbohydrate degradation. Importantly, in some species, GH161 and GH94 genes were present in tandem, providing evidence that GPs from different CAZy families may work sequentially to degrade oligosaccharides.
Project description:Glycoside phosphorylases catalyze the phosphorolysis of oligosaccharides into sugar phosphates. Recently, we found a novel phosphorylase acting on ?-1,2-glucooligosaccharides with degrees of polymerization of 3 or more (1,2-?-oligoglucan phosphorylase, SOGP) in glycoside hydrolase family (GH) 94. Here, we characterized SOGP from Lachnoclostridium phytofermentans (LpSOGP) and determined its crystal structure. LpSOGP is a monomeric enzyme that contains a unique ?-sandwich domain (Ndom1) at its N-terminus. Unlike the dimeric GH94 enzymes possessing catalytic pockets at their dimer interface, LpSOGP has a catalytic pocket between Ndom1 and the catalytic domain. In the complex structure of LpSOGP with sophorose, sophorose binds at subsites +1 to +2. Notably, the Glc moiety at subsite +1 is flipped compared with the corresponding ligands in other GH94 enzymes. This inversion suggests the great distortion of the glycosidic bond between subsites -1 and +1, which is likely unfavorable for substrate binding. Compensation for this disadvantage at subsite +2 can be accounted for by the small distortion of the glycosidic bond in the sophorose molecule. Therefore, the binding mode at subsites +1 and +2 defines the substrate specificity of LpSOGP, which provides mechanistic insights into the substrate specificity of a phosphorylase acting on ?-1,2-glucooligosaccharides.
Project description:Glycoside phosphorylases (GPs) with specificity for ?-(1???3)-gluco-oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far-glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X-ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on ?-(1???3)-gluco-oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.
Project description:?-Glucan phosphorylases contribute to degradation of glycogen and maltodextrins formed in the course of maltose metabolism in bacteria. Accordingly, bacterial ?-glucan phosphorylases are classified as either glycogen or maltodextrin phosphorylase, GlgP or MalP, respectively. GlgP and MalP enzymes follow the same catalytic mechanism, and thus their substrate spectra overlap; however, they differ in their regulation: GlgP genes are constitutively expressed and the enzymes are controlled on the activity level, whereas expression of MalP genes are transcriptionally controlled in response to the carbon source used for cultivation. We characterize here the modes of control of the ?-glucan phosphorylase MalP of the Gram-positive Corynebacterium glutamicum. In accordance to the proposed function of the malP gene product as MalP, we found transcription of malP to be regulated in response to the carbon source. Moreover, malP transcription is shown to depend on the growth phase and to occur independently of the cell glycogen content. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. Since the latter is considered a typical feature of GlgPs, we propose that C. glutamicum MalP acts as both maltodextrin and glycogen phosphorylase and, based on these findings, we question the current system for classification of bacterial ?-glucan phosphorylases.Bacterial ?-glucan phosphorylases have been classified conferring to their purpose as either glycogen or maltodextrin phosphorylases. We found transcription of malP in C. glutamicum to be regulated in response to the carbon source, which is recognized as typical for maltodextrin phosphorylases. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. The latter is considered a typical feature of GlgPs. These findings, taken together, suggest that C. glutamicum MalP is the first ?-glucan phosphorylase that does not fit into the current system for classification of bacterial ?-glucan phosphorylases and exemplifies the complex mechanisms underlying the control of glycogen content and maltose metabolism in this model organism.
Project description:In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.
Project description:Family GH13_18 of the carbohydrate-active enzyme database consists of retaining glycoside phosphorylases that have attracted interest with their potential for synthesizing valuable ?-sugars and glucosides. Sucrose phosphorylase was believed to be the only enzyme with specificity in this subfamily for many years, but recent work revealed an enzyme with a different function and hinted at an even broader diversity that is left to discover. In this study, a putative sucrose phosphorylase from Meiothermus silvanus that resides in a previously unexplored branch of the family's phylogenetic tree was expressed and characterized. Unexpectedly, no activity on sucrose was observed. Guided by a thorough inspection of the genomic landscape surrounding other genes in the branch, the enzyme was found to be a glucosylglycerate phosphorylase, with a specificity never before reported. Homology modeling, docking, and mutagenesis pinpointed particular acceptor site residues (Asn275 and Glu383) involved in the binding of glycerate. Various organisms known to synthesize and accumulate glucosylglycerate as a compatible solute possess a putative glucosylglycerate phosphorylase gene, indicating that the phosphorylase may be a regulator of its intracellular levels. Moreover, homologs of this novel enzyme appear to be distributed among diverse bacterial phyla, a finding which suggests that many more organisms may be capable of assimilating or synthesizing glucosylglycerate than previously assumed.IMPORTANCE Glycoside phosphorylases are an intriguing group of carbohydrate-active enzymes that have been used for the synthesis of various economically appealing glycosides and sugars, and they are frequently subjected to enzyme engineering to further expand their application potential. The novel specificity discovered in this work broadens the diversity of these phosphorylases and opens up new possibilities for the efficient production of glucosylglycerate, which is a remarkably potent and versatile stabilizer for protein formulations. Finally, it is a new piece of the puzzle of glucosylglycerate metabolism, being the only known enzyme capable of catalyzing the breakdown of glucosylglycerate in numerous bacterial phyla.
Project description:Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl ?-d-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenyl from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining ?-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to N-acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying ?-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications.
Project description:Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.
Project description:?-1,3-Glucan is present as the outermost layer of the cell wall in the pathogenic yeastlike (Y) form of Paracoccidioides brasiliensis. Based on experimental evidence, this polysaccharide has been proposed as a fungal virulence factor. To degrade ?-1,3-glucan and allow remodeling of the cell wall, ?-1,3-glucanase is required. Therefore, the study of this enzyme, its encoding gene, and regulatory mechanisms, might be of interest to understand the morphogenesis and virulence process in this fungus. A single gene, orthologous to other fungal ?-1,3-glucanase genes, was identified in the Paracoccidioides genome, and labeled AGN1. Transcriptional levels of AGN1 and AGS1 (?-1,3-glucan synthase-encoding gene) increased sharply when the pathogenic Y phase was cultured in the presence of 5% horse serum, a reported booster for cell wall ?-1,3-glucan synthesis in this fungus. To study the biochemical properties of P. brasiliensis Agn1p, the enzyme was heterologously overexpressed, purified, and its activity profile determined by means of the degradation of carboxymethyl ?-1,3-glucan (SCMG, chemically modified from P. brasiliensis ?-1,3-glucan), used as a soluble substrate for the enzymatic reaction. Inhibition assays, thin layer chromatography and enzymatic reactions with alternative substrates (dextran, starch, chitin, laminarin and cellulose), showed that Agn1p displays an endolytic cut pattern and high specificity for SCMG. Complementation of a Schizosaccharomyces pombe agn1? strain with the P. brasiliensis AGN1 gene restored the wild type phenotype, indicating functionality of the gene, suggesting a possible role of Agn1p in the remodeling of P. brasiliensis Y phase cell wall. Based on amino acid sequence, P. brasiliensis Agn1p, groups within the family 71 of fungal glycoside hydrolases (GH-71), showing similar biochemical characteristics to other members of this family. Also based on amino acid sequence alignments, we propose a subdivision of fungal GH-71 into at least five groups, for which specific conserved sequences can be identified.
Project description:The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-?-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for ?-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed ?-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (?-1,3-/?-1,4-) glucans such as barley ?-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the ?-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.