Functional Analyses of Resurrected and Contemporary Enzymes Illuminate an Evolutionary Path for the Emergence of Exolysis in Polysaccharide Lyase Family 2

McLean, Richard; Hobbs, Joanne K.; Suits, M.D.L.; Tuomivaara, Sami T.; Jones, Darryl R.; Boraston, A.B.; Abbott, D. Wade

doi:10.1074/jbc.m115.664847

Cited by 12 publications

(6 citation statements)

References 48 publications

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“…To demonstrate the utility of the TogB D363C-MDCC biosensor for characterizing enzyme activity, we examined oligogalacturonide release from the CAZyme-catalyzed degradation of PGA in real time. YePL2b is an exo-acting polysaccharide lyase from Y. enterocolitica that cleaves PGA using β–elimination, producing unsatdigalUA as the major product [ 33 , 35 ]. Using the stopped-flow method, a solution containing TogB D363C-MDCC and YePL2b was rapidly mixed with PGA, and the resulting fluorescent time courses were recorded and best fit with a two-exponential function ( Figure 4 , Equation (6)).…”

Section: Resultsmentioning

confidence: 99%

“…Overexpression and purification of YePL2b and YeGH28. The overexpression of YePL2b and YeGH28 was achieved using the same procedure as the overexpression of TogB variants, except the final concentration of IPTG used for induction was 200 µM [ 33 ]. The purification procedure for YePL2b and YeGH28 was the same as the purification of TogB variants, except fractions in Buffer C were buffer exchanged into Buffer E (20 mM Tris-Cl (pH 8.0 @ 20 °C)) using dialysis (30 mL sample in 500 mL Buffer E, 4 changes, molecular weight cut-off 12.4 kDa Sigma PN: D0530).…”

Section: Methodsmentioning

confidence: 99%

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Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform

Smith

King

Abbott

et al. 2022

Sensors

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Fluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a “Computational Identification of Non-disruptive Conjugation sites” (CINC) approach, an in silico pipeline utilizing molecular dynamics simulations for the rapid design and construction of novel protein–fluorophore conjugate-type biosensors. Here, we report an improved in silico scoring algorithm for use in CINC and its use in the construction of an oligogalacturonide-detecting biosensor set. Using both 4,5-unsaturated and saturated oligogalacturonides, we demonstrate that signal transmission from the ligand-binding pocket of the starting protein scaffold to the CINC-selected reporter positions is effective for multiple different ligands. The utility of an oligogalacturonide-detecting biosensor is shown in Carbohydrate Active Enzyme (CAZyme) activity assays, where the biosensor is used to follow product release upon polygalacturonic acid (PGA) depolymerization in real time. The oligogalacturonide-detecting biosensor set represents a novel enabling tool integral to our rapidly expanding platform for biosensor-based carbohydrate detection, and moving forward, the CINC pipeline will continue to enable the rational design of biomolecular tools to detect additional chemically distinct oligosaccharides and other solutes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform

Smith

King

Abbott

et al. 2022

Sensors

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“…Expression of BgaA and GH20C has been reported previously (16,30). Standard procedures, as detailed previously (62), were used to lyse cells and purify the released proteins by immobilized metal-affinity chromatography and size-exclusion chromatography using either an S200 or S300 HiPrep 16/60 Sephacryl column (GE Healthcare) as appropriate. Protein purity was judged by SDS-PAGE analysis, and protein concentrations were determined using extinction coefficients calculated by ProtParam on the ExPASy server (63).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Two complementary α-fucosidases from Streptococcus pneumoniae promote complete degradation of host-derived carbohydrate antigens

Hobbs

Pluvinage

Robb

et al. 2019

Journal of Biological Chemistry

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An important aspect of the interaction between the opportunistic bacterial pathogen Streptococcus pneumoniae and its human host is its ability to harvest host glycans. The pneumococcus can degrade a variety of complex glycans, including Nand O-linked glycans, glycosaminoglycans, and carbohydrate antigens, an ability that is tightly linked to the virulence of S. pneumoniae. Although S. pneumoniae is known to use a sophisticated enzyme machinery to attack the human glycome, how it copes with fucosylated glycans, which are primarily histoblood group antigens, is largely unknown. Here, we identified two pneumococcal enzymes, SpGH29 C and SpGH95 C , that target ␣-(133/4) and ␣-(132) fucosidic linkages, respectively. X-ray crystallography studies combined with functional assays revealed that SpGH29 C is specific for the Lewis A and Lewis X antigen motifs and that SpGH95 C is specific for the H(O)-antigen motif. Together, these enzymes could defucosylate Lewis Y and Lewis B antigens in a complementary fashion. In vitro reconstruction of glycan degradation cascades disclosed that the individual or combined activities of these enzymes expose the underlying glycan structure, promoting the complete deconstruction of a glycan that would otherwise be resistant to pneumococcal enzymes. These experiments expand our understanding of the extensive capacity of S. pneumoniae to process host glycans and the likely roles of ␣-fucosidases in this. Overall, given the importance of enzymes that initiate glycan breakdown in pneumococcal virulence, such as the neuraminidase NanA and the mannosidase SpGH92, we anticipate that the ␣-fucosidases identified here will be important factors in developing more refined models of the S. pneumoniae-host interaction. This work was supported by Canadian Institutes of Health Research Operating Grant PJT 159786. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S4 and Table S1. The atomic coordinates and structure factors (codes 6ORG, 6ORF, 6ORH, and 6OR4) have been deposited in the Protein Data Bank (http://wwpdb.org/).

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“…Glu 155 and Glu 254 in NF2152 were predicted to be the acid/ base and nucleophile, respectively, based upon superimposition with the GH51 from T. maritima (3UG4) (34). These residues were targeted for substitution to glutamine using site-directed mutagenesis with pET28_nf2152_29 -431 for template as previously described (68). Mutations were confirmed by DNA sequencing (Eurofins Genomics).…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

Discovery and characterization of family 39 glycoside hydrolases from rumen anaerobic fungi with polyspecific activity on rare arabinosyl substrates

Jones

Uddin

Gruninger

et al. 2017

Journal of Biological Chemistry

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Enzyme activities that improve digestion of recalcitrant plant cell wall polysaccharides may offer solutions for sustainable industries. To this end, anaerobic fungi in the rumen have been identified as a promising source of novel carbohydrate active enzymes (CAZymes) that modify plant cell wall polysaccharides and other complex glycans. Many CAZymes share insufficient sequence identity to characterized proteins from other microbial ecosystems to infer their function; thus presenting challenges to their identification. In this study, four rumen fungal genes (, ,, and ) were identified that encode family 39 glycoside hydrolases (GH39s), and have conserved structural features with GH51s. Two recombinant proteins, NF2152 and NF2523, were characterized using a variety of biochemical and structural techniques, and were determined to have distinct catalytic activities. NF2152 releases a single product, β1,2-arabinobiose (Ara) from sugar beet arabinan (SBA), and β1,2-Ara and α-1,2-galactoarabinose (Gal-Ara) from rye arabinoxylan (RAX). NF2523 exclusively releases α-1,2-Gal-Ara from RAX, which represents the first description of a galacto-(α-1,2)-arabinosidase. Both β-1,2-Ara and α-1,2-Gal-Ara are disaccharides not previously described within SBA and RAX. In this regard, the enzymes studied here may represent valuable new biocatalytic tools for investigating the structures of rare arabinosyl-containing glycans, and potentially for facilitating their modification in industrial applications.

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Functional Analyses of Resurrected and Contemporary Enzymes Illuminate an Evolutionary Path for the Emergence of Exolysis in Polysaccharide Lyase Family 2

Cited by 12 publications

References 48 publications

Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform

Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform

Two complementary α-fucosidases from Streptococcus pneumoniae promote complete degradation of host-derived carbohydrate antigens

Discovery and characterization of family 39 glycoside hydrolases from rumen anaerobic fungi with polyspecific activity on rare arabinosyl substrates

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