Structure of<i>Aspergillus aculeatus</i>β-1,4-galactanase in complex with galactobiose

Torpenholt, Søs; Poulsen, J.C. Navarro; Muderspach, Sebastian J.; Maria, Leonardo De; Leggio, Leila Lo

doi:10.1107/s2053230x19005612

Cited by 6 publications

(4 citation statements)

References 27 publications

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“…As expected, the fungal galactanases were able to degrade G3. The accumulation of G1 and G2 is also expected and supported by an X-ray crystal structure of the fungal galactanase AaGal binding galactobiose unproductively at the − 1 and − 2 binding subsites [ 23 ]. The pattern of IaGal therefore proved highly distinct from the fungal galactanases as it was not able to degrade G3.…”

Section: Discussionmentioning

confidence: 80%

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Engineering the substrate binding site of the hyperthermostable archaeal endo-β-1,4-galactanase from Ignisphaera aggregans

Muderspach

Fredslund

Volf

et al. 2021

Biotechnol Biofuels

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Background Endo-β-1,4-galactanases are glycoside hydrolases (GH) from the GH53 family belonging to the largest clan of GHs, clan GH-A. GHs are ubiquitous and involved in a myriad of biological functions as well as being widely used industrially. Endo-β-1,4-galactanases, in particular hydrolyse galactan and arabinogalactan in pectin, a major component of the primary plant cell wall, with important functions in plant defence and application in the food and other industries. Here, we explore the family’s biological diversity by characterizing the first archaeal and hyperthermophilic GH53 galactanase, and utilize it as a scaffold for engineering enzymes with different product lengths. Results A galactanase gene was identified in the genome of the anaerobic hyperthermophilic archaeon Ignisphaera aggregans, and the isolated catalytic domain expressed and characterized (IaGal). IaGal presents the typical (βα)8 barrel structure of clan GH-A enzymes, with catalytic carboxylates at the end of the 4th and 7th barrel strands. Its activity optimum of at least 95 °C and melting point over 100 °C indicate extreme thermostability, a very advantageous property for industrial applications. If enzyme depletion is reduced, so is the need for re-addition, and thus costs. The main stabilizing features of IaGal compared to other structurally characterized members are π–π and cation–π interactions. The length of the substrate binding site—and thus produced oligosaccharide products—is intermediate compared to previously characterized galactanases. Variants inspired by the structural diversity in the GH53 family were rationally designed to shorten or extend the substrate binding groove, in order to modulate product length. Subsite-deleted variants produced shorter products than IaGal, as do the fungal galactanases inspiring the design. IaGal variants engineered with a longer binding site produced a less expected degradation pattern, though still different from that of wild-type IaGal. All variants remained extremely stable. Conclusions We have characterized in detail the most thermophilic endo-β-1,4-galactanase known to date and successfully engineered it to modify the degradation profile, while maintaining much of its desirable thermostability. This is an important achievement as oligosaccharide products length is an important property for industrial and natural GHs alike.

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Section: Discussionmentioning

confidence: 80%

“…A structure of AaGal in complex with galactobiose has been determined (PDB ID 6Q3R), elucidating binding at the − 1 and − 2 subsites [ 23 ]. This binding mode agrees with the observed degradation patterns of fungal galactanases seen in Ryttersgaard et al .…”

Section: Introductionmentioning

confidence: 99%

Engineering the substrate binding site of the hyperthermostable archaeal endo-β-1,4-galactanase from Ignisphaera aggregans

Muderspach

Fredslund

Volf

et al. 2021

Biotechnol Biofuels

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“…Outside of B-group blood antigen binders, PDB entries with consecutive galactose residues are infrequent and don’t always employ the same α1-3 linkage as found in α-gal, although most involve a tryptophan–GAL interface ( SI Appendix , Table S8 ); however, unlike the antibody structures, most employ a small binding pocket accommodating just the terminal (proximal) sugar unit (usually GAL, but sometimes GLA). In the cases of some glycosyl hydrolases, two or three consecutive GAL residues of poly-GAL substrates are captured in the substrate entry channels, each similarly interfaced with its own tryptophan residue [PDB entries 6q3r ( 46 ) and 2ccr ( 47 ), respectively].…”

Section: Discussionmentioning

confidence: 99%

Genetic and structural basis of the human anti-α-galactosyl antibody response

Langley

Schofield

Névoltris

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Humans lack the capacity to produce the Galα1–3Galβ1–4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.

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“…This group of enzymes can be divided into β-1,3-galactanase, β-1,4-galactanase and β-1,6-galactanase according to the structure of their substrates [ 13 ]. At present, there are many studies on the preparation and functional analysis of β-1,4-galactanase, whereas investigations with β-1,3- and β-1,6-galactanases are relatively rare [ 14 , 15 ]. Therefore, it has become crucial to the field to prepare and characterize β-1,3- and β-1,6-galactanases.…”

Section: Introductionmentioning

confidence: 99%

Molecular Cloning and Characterization of a Novel Exo-β-1,3-Galactanase from Penicillium oxalicum sp. 68

Zhou

Yan

et al. 2022

J. Microbiol. Biotechnol.

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β-D-(1→4)-galactan, whereas AG-II is β-D-(1→3) and/or (1→6)-galactan. Large quantities of AGs are present in Larix trees [1][2][3], and have been shown to possess diverse biological properties, including immunological activity [4], antitumor [5], and antiviral effects [6]. AGs from Larix laricina have been reported to play a unique role in reducing the incidence of the common cold [7]. In addition, AGs have been approved by the US Food and Drug Administration (FDA) for use as dietary fiber. AG-mediated biological activity is generally associated with its monosaccharide composition, type of glycosidic linkage, molecular weight, as well as the number and type of substituents and branched chains. Therefore, analysis of the complex fine structure and function is a significant problem in glycobiology.Glycoside hydrolase is an important enzyme that is widely used in the study of polysaccharides [8]. Up to now, about 171 glycoside hydrolase families have been reported in the CAZy database [9]. β-Galactanases are a broad group of enzymes that hydrolyze glycosidic bonds in plant-derived galactans, and have been widely used in the analysis of polysaccharide structures, construction of medicinal plant fingerprints, preparation of galactooligosaccharides, and food quality improvement [10][11][12]. This group of enzymes can be divided into β-1,3-galactanase, β-1,4galactanase and β-1,6-galactanase according to the structure of their substrates [13]. At present, there are many studies on the preparation and functional analysis of β-1,4-galactanase, whereas investigations with β-1,3-and β-1,6-galactanases are relatively rare [14,15]. Therefore, it has become crucial to the field to prepare and characterize β-1,3-and β-1,6-galactanases. Arabinogalactans have diverse biological properties and can be used as pharmaceutical agents.Most arabinogalactans are composed of β-(1 → 3)-galactan, so it is particularly important to identify β-1,3-galactanases that can selectively degrade them. In this study, a novel exo-β-1,3-galactanase, named PoGal3, was screened from Penicillium oxalicum sp. 68, and hetero-expressed in P. pastoris GS115 as a soluble protein. PoGal3 belongs to glycoside hydrolase family 43 (GH43) and has a 1,356bp gene length that encodes 451 amino acids residues. To study the enzymatic properties and substrate selectivity of PoGal3, β-1,3-galactan (AG-P-I) from larch wood arabinogalactan (LWAG) was prepared and characterized by HPLC and NMR. Using AG-P-I as substrate, purified PoGal3 exhibited an optimal pH of 5.0 and temperature of 40°C. We also discovered that Zn 2+ had the strongest promoting effect on enzyme activity, increasing it by 28.6%. Substrate specificity suggests that PoGal3 functions as an exo-β-1,3-galactanase, with its greatest catalytic activity observed on AG-P-I. Hydrolytic products of AG-P-I are mainly composed of galactose and β-1,6-galactobiose. In addition, PoGal3 can catalyze hydrolysis of LWAG to produce galacto-oligomers. PoGal3 is the first enzyme identified as an exo-β-1,3-galactanase that can b...

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Structure ofAspergillus aculeatusβ-1,4-galactanase in complex with galactobiose

Cited by 6 publications

References 27 publications

Engineering the substrate binding site of the hyperthermostable archaeal endo-β-1,4-galactanase from Ignisphaera aggregans

Engineering the substrate binding site of the hyperthermostable archaeal endo-β-1,4-galactanase from Ignisphaera aggregans

Genetic and structural basis of the human anti-α-galactosyl antibody response

Molecular Cloning and Characterization of a Novel Exo-β-1,3-Galactanase from Penicillium oxalicum sp. 68

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