| 1. |
- Aurelius, Oskar, et al.
(författare)
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The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:7
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.
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| 2. |
- Bågenholm, Viktoria, et al.
(författare)
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A surface-exposed GH26 -mannanase from Bacteroides ovatus : Structure, role, and phylogenetic analysis of BoMan26B
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 294:23, s. 9100-9117
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Tidskriftsartikel (refereegranskat)abstract
- The galactomannan utilization locus (BoManPUL) of the human gut bacterium Bacteroides ovatus encodes BoMan26B, a cell-surface– exposed endomannanase whose functional and structural features have been unclear. Our study now places BoMan26B in context with related enzymes and reveals the structural basis for its specificity. BoMan26B prefers longer substrates and is less restricted by galactose side-groups than the mannanase BoMan26A of the same locus. Using galactomannan, BoMan26B generated a mixture of (galactosyl) manno-oligosaccharides shorter than mannohexaose. Three defined manno-oligosaccharides had affinity for the SusD-like surface–exposed glycan-binding protein, predicted to be implicated in saccharide transport. Co-incubation of BoMan26B and the periplasmic -galactosidase BoGal36A increased the rate of galactose release by about 10-fold compared with the rate without BoMan26B. The results suggested that BoMan26B performs the initial attack on galactomannan, generating oligosaccharides that after transport to the periplasm are processed by BoGal36A. A crystal structure of BoMan26B with galactosyl-mannotetraose bound in subsites 5 to 2 revealed an open and long active-site cleft with Trp-112 in subsite 5 concluded to be involved in mannosyl interaction. Moreover, Lys-149 in the 4 subsite interacted with the galactosyl side-group of the ligand. A phylogenetic tree consisting of GH26 enzymes revealed four strictly conserved GH26 residues and disclosed that BoMan26A and BoMan26B reside on two distinct phylogenetic branches (A and B). The three other branches contain lichenases, xylanases, or enzymes with unknown activities. Lys-149 is conserved in a narrow part of branch B, and Trp-112 is conserved in a wider group within branch B.
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| 3. |
- Bågenholm, Viktoria, et al.
(författare)
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Galactomannan catabolism conferred by a polysaccharide utilisation locus of Bacteroides ovatus : enzyme synergy and crystal structure of a β-mannanase
- 2017
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 292:1, s. 229-243
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Tidskriftsartikel (refereegranskat)abstract
- A recently identified polysaccharide utilization locus (PUL) from Bacteroides ovatus ATCC 8483 is transcriptionally up-regulated during growth on galacto- and glucomannans. It encodes two glycoside hydrolase family 26 (GH26) β-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan-binding proteins, confirmed by β-mannan affinity electrophoresis. When this PUL was deleted, B. ovatus was no longer able to grow on locust bean galactomannan. BoMan26A primarily formed mannobiose from mannan polysaccharides. BoMan26B had higher activity on galactomannan with a high degree of galactosyl substitution and was shown to be endo-acting generating a more diverse mixture of oligosaccharides, including mannobiose. Of the two β-mannanases, only BoMan26B hydrolyzed galactoglucomannan. A crystal structure of BoMan26A revealed a similar structure to the exo-mannobiohydrolase CjMan26C from Cellvibrio japonicus, with a conserved glycone region (-1 and -2 subsites), including a conserved loop closing the active site beyond subsite -2. Analysis of cellular location by immunolabeling and fluorescence microscopy suggests that BoMan26B is surface-exposed and associated with the outer membrane, although BoMan26A and BoGal36A are likely periplasmic. In light of the cellular location and the biochemical properties of the two characterized β-mannanases, we propose a schemeof sequential action by the glycoside hydrolasesencodedby the β-mannanPULandinvolved in the β-mannanutilization pathway in B. ovatus. The outer membrane-associated BoMan26B initially acts on the polysaccharide galactomannan, producing comparably large oligosaccharide fragments. Galactomanno-oligosaccharides are further processed in the periplasm, degalactosylated by BoGal36A, and subsequently hydrolyzed into mainly mannobiose by the β-mannanase BoMan26A.
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| 4. |
- Bågenholm, Viktoria
(författare)
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The digestive machinery of a human gut bacterium : Structural enzymology of galactomannan utilisation
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human gut bacteria utilise different types of polysaccharides present in our diet. One of these polysaccharides is galactomannan. Many organisms in the phylum Bacteroidetes have gene clusters encoding for all proteins required for hydrolysis, binding and transport of one type of polysaccharide, called polysaccharide utilisation loci (PULs). A PUL from the human gut bacterium Bacteroides ovatus was previously indicated to be involved in galactomannan utilisation (BoManPUL) and codes for one glycosde hydrolase (GH) family 36 α-galactosidase (BoGal36A, Paper I) and two GH26 β-mannanases (BoMan26A and BoMan26B, Papers II-IV). In Paper I B. ovatus was shown to be able to grow on galactomannan, an ability which was lost upon knockout of BoManPUL, indicating that it was primarily responsible for galactomanna utilisation in this human gut bacteria. Papers I-III revealed a pathway for galactomanna utilisation in which BoMan26B initially cleaves polysaccharide substrates outside the cell, the products of which are transported into the periplasm and there further processed by BoGal36A, then BoMan26A. BoMan26B is outer membrane attached, preferntially hydrolyses longer substrates (Paper II) and is one of the enzymes in GH26 least restricted by galactose substitutions (Papers II and III). Crystal structures revealed a long, open active site cleft, only being restricted by galactose substitutions in one subsite -2 and possibly favouring a substitution in subsite -4 (Paper III). BoMan26B provides sequential synergy to BoGal36A (Paper III), which preferentially hydrolyses internal galactose substitutions from galactomanno-oligosaccharides (Paper I). This hydrolysis of internal galactosyl units is unusual in GH36, which was shown to be caused by the absence of a loop that is present in other GH36 subgroup I members (Paper I). BoGal36A in turn provides sequential synergy to the endo-capable mannobiohydrolase BoMan26A, which preferentially cleaves unsubstituted mannooligosaccharides (Paper II). The structure of BoMan26A revealed a narrow active site cleft where at least two subsites are restricted by galactose substitutions, and which was blocked beyond subsite -2 by two loops: 2 and 8 (Paper II). NMR assignment of the BoMan26A backbone was carried out in Paper IV, to perform further NMR-based studies of substarte binding and protein dynamics relating to loops 2 and 8. BoMan26A and BoMan26B differ both in structure and biochemistry and in a phylogenetic analysis of selected GH26 sequences they were shown to cluster in different branches (Paper III). The level of conservation around the active site cleft was generally low, with the exceptions of the -1 and +1 subsites (Paper III). This thesis reveals a model for galactomannan PUL utilisation in B. ovatus, increasing the understanding of our gut microbiota. It also delves into the structure-function relationship of substrate specificity in, primarily, GH26 enzymes.
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| 5. |
- Grønberg, Christina, et al.
(författare)
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Structure and ion-release mechanism of P IB-4-type ATPases
- 2022
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Ingår i: eLife. - : eLife Sciences Publications Ltd. - 2050-084X.
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Tidskriftsartikel (refereegranskat)abstract
- Abstract Transition metals, such as zinc, are essential micronutrients in all organisms, but alsohighly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial forhomeostasis, conferring cellular detoxification and redistribution through transport of these ionsacross cellular membranes. No structural information is available for the PIB-4-ATPases, the subclasswith the broadest cargo scope, and hence even their topology remains elusive. Here, we presentstructures and complementary functional analyses of an archetypal PIB-4-ATPase, sCoaT fromSulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy-metal-binding domains (HMBDs), and provide fundamentally new insights into the mechanism anddiversity of heavy-metal transporters. We reveal several novel P-type ATPase features, includinga dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We alsoestablish that the turnover of PIB-ATPases is potassium independent, contrasting to many otherP-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in forexample drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.
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| 6. |
- Guo, Zongxin, et al.
(författare)
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Diverse roles of the metal binding domains and transport mechanism of copper transporting P-type ATPases
- 2024
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Copper transporting P-type (P1B-1-) ATPases are essential for cellular homeostasis. Nonetheless, the E1-E1P-E2P-E2 states mechanism of P1B-1-ATPases remains poorly understood. In particular, the role of the intrinsic metal binding domains (MBDs) is enigmatic. Here, four cryo-EM structures and molecular dynamics simulations of a P1B-1-ATPase are combined to reveal that in many eukaryotes the MBD immediately prior to the ATPase core, MBD−1, serves a structural role, remodeling the ion-uptake region. In contrast, the MBD prior to MBD−1, MBD−2, likely assists in copper delivery to the ATPase core. Invariant Tyr, Asn and Ser residues in the transmembrane domain assist in positioning sulfur-providing copper-binding amino acids, allowing for copper uptake, binding and release. As such, our findings unify previously conflicting data on the transport and regulation of P1B-1-ATPases. The results are critical for a fundamental understanding of cellular copper homeostasis and for comprehension of the molecular bases of P1B-1-disorders and ongoing clinical trials.
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| 7. |
- Krishnaswamyreddy, Sumitha, et al.
(författare)
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A β-mannan utilisation locus in Bacteroides ovatus involves a GH36 α-galactosidase active on galactomannans
- 2016
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 590:14, s. 2106-2118
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Tidskriftsartikel (refereegranskat)abstract
- The Bacova_02091 gene in the β-mannan utilisation locus of Bacteroides ovatus encodes a family GH36 α-galactosidase (BoGal36A), transcriptionally upregulated during growth on galactomannan. Characterisation of recombinant BoGal36A reveals unique properties compared to other GH36 α-galactosidases, which preferentially hydrolyse terminal α-galactose in raffinose family oligosaccharides. BoGal36A prefers hydrolysing internal galactose substitutions from intact and depolymerized galactomannan. BoGal36A efficiently releases (>90%) galactose from guar and locust bean galactomannans, resulting in precipitation of the polysaccharides. As compared to other GH36 structures, the BoGal36A 3D model displays a loop deletion, resulting in a wider active site cleft which likely can accommodate a galactose-substituted polymannose backbone. This article is protected by copyright. All rights reserved.
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| 8. |
- Wernersson, Sven, et al.
(författare)
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Backbone H-1, C-13, and N-15 resonance assignments of BoMan26A, a -mannanase of the glycoside hydrolase family 26 from the human gut bacterium Bacteroides ovatus
- 2019
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Ingår i: Biomolecular NMR Assignments. - : Springer Science and Business Media LLC. - 1874-2718 .- 1874-270X. ; 13:1, s. 213-218
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Tidskriftsartikel (refereegranskat)abstract
- Bacteroides ovatus is a member of the human gut microbiota. The importance of this microbial consortium involves the degradation of complex dietary glycans mainly conferred by glycoside hydrolases. In this study we focus on one such catabolic glycoside hydrolase from B. ovatus. The enzyme, termed BoMan26A, is a -mannanase that takes part in the hydrolytic degradation of galactomannans. The crystal structure of BoMan26A has previously been determined to reveal a TIM-barrel like fold, but the relation between the protein structure and the mode of substrate processing has not yet been studied. Here we report residue-specific assignments for 95% of the 344 backbone amides of BoMan26A. The assignments form the basis for future studies of the relationship between substrate interactions and protein dynamics. In particular, the potential role of loops adjacent to glycan binding sites is of interest for such studies.
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