| 1. |
- Becker, D., et al.
(författare)
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Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum : the pH behaviour of Trichoderma reesei CeI7A and its E223S/A224H/L225V/T226A/D262G mutant
- 2001
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 356, s. 19-30
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves, The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Angstrom (= 0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 Angstrom contact between N-2 and O'(1). The pH variation of k(cat)/K-m for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wildtype and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K-m values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced nonproductive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.
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| 2. |
- Chang, Shu-Chieh, et al.
(författare)
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The Gram-positive bacterium Romboutsia ilealis harbors a polysaccharide synthase that can produce (1,3;1,4)-β-D-glucans
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- (1,3;1,4)-β-D-Glucans are widely distributed in the cell walls of grasses (family Poaceae) and closely related families, as well as some other vascular plants. Additionally, they have been found in other organisms, including fungi, lichens, brown algae, charophycean green algae, and the bacterium Sinorhizobium meliloti. Only three members of the Cellulose Synthase-Like (CSL) genes in the families CSLF, CSLH, and CSLJ are implicated in (1,3;1,4)-β-D-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-β-D-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-β-D-glucans in the exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis CRIBT. We also report that RiGT2 is the candidate gene of R. ilealis that encodes (1,3;1,4)-β-D-glucan synthase. RiGT2 has conserved glycosyltransferase family 2 (GT2) motifs, including D, D, D, QXXRW, and a C-terminal PilZ domain that resembles the C-terminal domain of bacteria cellulose synthase, BcsA. Using a direct gain-of-function approach, we insert RiGT2 into Saccharomyces cerevisiae, and (1,3;1,4)-β-D-glucans are produced with structures similar to those of the (1,3;1,4)-β-D-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-β-D-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-β-D-glucans in these species.
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| 5. |
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| 7. |
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| 8. |
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| 10. |
- Furlanetto, Valentina, et al.
(författare)
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LolA and LolB from the plant-pathogen Xanthomonas campestris forms a stable heterodimeric complex in the absence of lipoprotein
- 2023
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- The Gram-negative bacterium Xanthomonas campestris is one of the most problematic phytopathogens, and especially the pathovar campestris (Xcc) that causes a devastating plant disease known as black rot and it is of considerable interest to understand the molecular mechanisms that enable virulence and pathogenicity. Gram-negative bacteria depend on lipoproteins (LPs) that serve many important functions including control of cell shape and integrity, biogenesis of the outer membrane (OM) and establishment of transport pathways across the periplasm. The LPs are localized to the OM where they are attached via a lipid anchor by a process known as the localization of lipoprotein (Lol) pathway. Once a lipid anchor has been synthesized on the nascent LP, the Lol pathway is initiated by a membrane-bound ABC transporter that extracts the lipid anchor of the LP from the IM. The ABC extractor presents the extracted LP to the transport protein LolA, which binds the anchor and thereby shields it from the hydrophilic periplasmic milieu. It is assumed that LolA then carries the LP across the periplasm to the OM. At the periplasmic face of the OM, the LP cargo is delivered to LolB, which completes the Lol pathway by inserting the LP anchor in the inner leaflet of the outer membrane. Earlier studies have shown that loss of Xcc LolA or LolB leads to decreased virulence and pathogenicity during plant infection, which motivates studies to better understand the Lol system in Xcc. In this study, we report the first experimental structure of a complex between LolA and LolB. The crystal structure reveals a stable LolA-LolB complex in the absence of LP. The structural integrity of the LP-free complex is safeguarded by specific protein-protein interactions that do not coincide with interactions predicted to participate in lipid binding. The results allow us to identify structural determinants that enable Xcc LolA to dock with LolB and initiate LP transfer.
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| 11. |
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| 12. |
- Furlanetto, Valentina, et al.
(författare)
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Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius
- 2024
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 436:9
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Tidskriftsartikel (refereegranskat)abstract
- Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+β subunits) that cleaves the glucosyl-aglycone C–C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2β2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C–C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min−1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min−1).
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| 13. |
- Furlanetto, Valentina
(författare)
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Symbiotic and pathogenic factors in plant-microbe interaction: Structural basis of C-glycoside metabolism and lipoprotein transport in bacteria
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The communication between plants and bacteria involves a complex chemical signaling network that mediates responses to various biotic and abiotic stresses, as well as establishing symbiotic relationships between different organisms.The first part of the thesis focuses on a mechanism for symbiotic communication between plants and bacteria and more specifically on how C-glycosylated aromatic polyketide compounds produced by plants can be used as a mechanism for plants to communicate with beneficial bacteria. In their glycosylated form, these compounds are substrates for symbiotic bacteria, which in return deglycosylate them and release the sugar-free part, the active aglycone. The aglycone can then mediate several functions beneficial to the plant, for example facilitating nitrogen fixation or acting as an antibacterial agent against plant pathogens.Results from studies covered in the thesis show that the soil bacteria Deinococcus aerius, Streptomyces canus and Microbacterium testaceum produce enzymes that can cleave the carbon-carbon bond between the sugar and the aglycone in C-glycosyl compounds. Deglycosylation first requires oxidation of the sugar by an oxidoreductase, after which the carbon-carbon bond can be cleaved by a C-glycosyl deglycosidase (CGD). Biochemical and structural characterization as well as results from phylogenetic analyzes of the amino-acid sequences of CGD enzymes provided new knowledge about these relatively unexplored enzymatic processes, as well as increased insight into how C-glycosylated aromatic polyketides participate in the interaction between plant and bacteria.The second part of the thesis explores pathogenic interactions between plants and bacteria. The virulence of pathogenic bacteria is dependent on lipoproteins that are attached to the bacteria's outer membrane and that have a decisive role for the bacteria's survival and pathogenicity. The localization of lipoproteins takes place through a process abbreviated Lol. The Lol system of the notorious plant pathogen Xanthomonas campestris was studied to better understand the underlying molecular mechanisms of the localization system, which could eventually open new ways to combat the bacterium. Biochemical, structural, and phylogenetic techniques were used also in this project.Taken together, the results contributed several new discoveries. For the first time, a physical complex between the two proteins responsible for transporting lipoproteins could be determined and their mutual interactions studied. Furthermore, sequence analyses challenge the generally accepted model of how lipoproteins are released from the bacterial inner membrane before being transported to the outer membrane. According to the standard model based on Escherichia coli, lipoproteins are extracted from the inner membrane by a membrane protein that belongs to the ABC-transporter family and whose structure forms an asymmetric heterodimer (LolCDE). However, our bioinformatic analysis show that most of these ABC transporters, including X. campestris, are likely to be homodimers and that Escherichia coli is the exception rather than the rule. The difference between an asymmetric and symmetric ABC transporter also has implications for several hypotheses about how these proteins function. Heterologous production of the X. campestris ABC transporter confirmed that the protein is a homodimer.
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| 14. |
- Gandini, Rosaria, et al.
(författare)
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A Transmembrane Crenarchaeal Mannosyltransferase Is Involved in N-Glycan Biosynthesis and Displays an Unexpected Minimal Cellulose-Synthase-like Fold
- 2020
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 432:16, s. 4658-4672
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Tidskriftsartikel (refereegranskat)abstract
- Protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms across all domains of life. A seemingly boundless number of enzymes, glycosyltransferases, are involved in the biosynthesis of these protein-linked glycans. Few glycanbiosynthetic glycosyltransferases have been characterized in vitro, mainly due to the majority being integral membrane proteins and the paucity of relevant acceptor substrates. The crenarchaeote Pyrobaculum calidifontis belongs to the TACK superphylum of archaea (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) that has been proposed as an eukaryotic ancestor. In archaea, N-glycans are mainly found on cell envelope surface-layer proteins, archaeal flagellins and pili. Archaeal N-glycans are distinct from those of eukaryotes, but one noteworthy exception is the high-mannose N-glycan produced by P. calidifontis, which is similar in sugar composition to the eukaryotic counterpart. Here, we present the characterization and crystal structure of the first member of a crenarchaeal membrane glycosyltransferase, PcManGT. We show that the enzyme is a GDP-, dolichylphosphate-, and manganese-dependent mannosyltransferase. The membrane domain of PcManGT includes three transmembrane helices that topologically coincide with "half' of the sixtransmembrane helix cellulose-binding tunnel in Rhodobacter spheroides cellulose synthase BcsA. Conceivably, this "half tunnel" would be suitable for binding the dolichylphosphate-linked acceptor substrate. The PcManGT gene (Pcal_0472) is located in a large gene cluster comprising 14 genes of which 6 genes code for glycosyltransferases, and we hypothesize that this cluster may constitute a crenarchaeal N-glycosylation (PNG) gene cluster.
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| 15. |
- Gandini, Rosaria, et al.
(författare)
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Structural basis for dolichylphosphate mannose biosynthesis
- 2017
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Protein glycosylation is a critical protein modification. In biogenic membranes of eukaryotes and archaea, these reactions require activated mannose in the form of the lipid conjugate dolichylphosphate mannose (Dol-P-Man). The membrane protein dolichylphosphate mannose synthase (DPMS) catalyzes the reaction whereby mannose is transferred from GDP-mannose to the dolichol carrier Dol-P, to yield Dol-P-Man. Failure to produce or utilize Dol-P-Man compromises organism viability, and in humans, several mutations in the human dpm1 gene lead to congenital disorders of glycosylation (CDG). Here, we report three high-resolution crystal structures of archaeal DPMS from Pyrococcus furiosus, in complex with nucleotide, donor, and glycolipid product. The structures offer snapshots along the catalytic cycle, and reveal how lipid binding couples to movements of interface helices, metal binding, and acceptor loop dynamics to control critical events leading to Dol-P-Man synthesis. The structures also rationalize the loss of dolichylphosphate mannose synthase function in dpm1-associated CDG.The generation of glycolipid dolichylphosphate mannose (Dol-P-Man) is a critical step for protein glycosylation and GPI anchor synthesis. Here the authors report the structure of dolichylphosphate mannose synthase in complex with bound nucleotide and donor to provide insight into the mechanism of Dol-P-Man synthesis.
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| 16. |
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| 17. |
- Hallberg, B. M., et al.
(författare)
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A new scaffold for binding haem in the cytochrome domain of the extracellular flavocytochrome cellobiose dehydrogenase
- 2000
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Ingår i: Structure. - 0969-2126 .- 1878-4186. ; 8:1, s. 79-88
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Tidskriftsartikel (refereegranskat)abstract
- Background: The fungal oxidoreductase cellobiose dehydrogenase (CDH) degrades both lignin and cellulose, and is the only known extracellular flavocytochrome. This haemoflavoenzyme has a multidomain organisation with a b-type cytochrome domain linked to a large flavodehydrogenase domain. The two domains can be separated proteolytically to yield a functional cytochrome and a flavodehydrogenase. Here, we report the crystal structure of the cytochrome domain of CDH. Results: The crystal structure of the b-type cytochrome domain of CDH from the wood-degrading fungus Phanerochaete chrysosporium has been determined at 1.9 Angstrom resolution using multiple isomorphous replacement ncluding anomalous scattering information. Three models of the cytochrome have been refined: the in vitro prepared cytochrome in its redox-inactive state (pH 7.5) and redox-active state (pH 4.6), as well as the naturally occurring cytochrome fragment. Conclusions: The 190-residue long cytochrome domain of CDH folds as a beta sandwich with the topology of the antibody Fab V-H domain. The haem iron is ligated by Met65 and His 163, which confirms previous results from spectroscopic studies. This is only the second example of a b-type cytochrome with this ligation, the first being cytochrome b(562). The haem-propionate groups are surface exposed and, therefore, might play a role in the association between the cytochrome and flavoprotein domain, and in interdomain electron transfer. There are no large differences in overall structure of the cytochrome at redoxactive pH as compared with the inactive form, which excludes the possibility that pH-dependent redox inactivation results from partial denaturation. From the electron-density map of the naturally occurring cytochrome, we conclude that it corresponds to the proteolytically prepared cytochrome domain.
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| 18. |
- Hallberg, B. M., et al.
(författare)
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Crystal structure of the flavoprotein domain of the extracellular flavocytochrome cellobiose dehydrogenase
- 2002
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 315:3, s. 421-434
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Tidskriftsartikel (refereegranskat)abstract
- Cellobiose dehydrogenase (CDH) participates in the degradation of cellulose and lignin. The protein is an extracellular flavocytochrome with a b-type cytochrome domain (CYTcdh) connected to a flavodehydrogenase domain (DHcdh). DHcdh catalyses a two-electron oxidation at the anomeric C1 position of cellobiose to yield cellobiono-1,5-lactone, and the electrons are subsequently transferred from DHcdh to an acceptor, either directly or via CYTcdh. Here, we decribe the crystal structure of Phanerochaete chrysosporium DHcdh determined at 1.5 Angstrom resolution. DHcdh belongs to the GMC family of oxidoreductases, which includes glucose oxidase (GOX) and cholesterol oxidase (COX); however, the sequence identity with members of the family is low. The overall fold of DHcdh is p-hydroxybenzoate hydroxylase-like and is similar to, but also different from, that of GOX and COX. It is partitioned into an FAD-binding subdomain of alpha/beta type and a substrate-binding subdomain consisting of a seven-stranded beta sheet and six helices. Docking of CYTcdh, and DHcdh suggests that CYTcdh covers the active-site entrance in DHcdh, and that the resulting distance between the cofactors is within acceptable limits for inter-domain electron transfer. Based on docking of the substrate, cellobiose, in the active site of DHcdh, we propose that the enzyme discriminates against glucose by favouring interaction with the non-reducing end of cellobiose.
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| 19. |
- Hallberg, B. M., et al.
(författare)
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Mechanism of the reductive half-reaction in cellobiose dehydrogenase
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:9, s. 7160-7166
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Tidskriftsartikel (refereegranskat)abstract
- The extracellular flavocytochrome cellobiose dehydrogenase (CDH; EC 1.1.99.18) participates in lignocellulose degradation by white-rot fungi with a proposed role in the early events of wood degradation. The complete hemoflavoenzyme consists of a catalytically active dehydrogenase fragment (DHcdh) connected to a b-type cytochrome domain via a linker peptide. In the reductive half-reaction, DHcdh catalyzes the oxidation of cellobiose to yield cellobiono-1,5-lactone. The active site of DHcdh is structurally similar to that of glucose oxidase and cholesterol oxidase, with a conserved histidine residue positioned at the re face of the flavin ring close to the N5 atom. The mechanisms of oxidation in glucose oxidase and cholesterol oxidase are still poorly understood, partly because of lack of experimental structure data or difficulties in interpreting existing data for enzyme-ligand complexes. Here we report the crystal structure of the Phanerochaete chrysosporium DHcdh with a bound inhibitor, cellobiono-1,5-lactam, at 1.8-Angstrom resolution. The distance between the lactam C1 and the flavin N5 is only 2.9 Angstrom, implying that in an approximately planar transition state, the maximum distance for the axial 1-hydrogen to travel for covalent addition to N5 is 0.8-0.9 Angstrom. The lactam O1 interacts intimately with the side chains of His-689 and Asn-732. Our data lend substantial structural support to a reaction mechanism where His-689 acts as a general base by abstracting the O1 hydroxyl proton in concert with transfer of the C1 hydrogen as hydride to the re face of the flavin N5.
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| 20. |
- Hallberg, Martin, et al.
(författare)
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Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase
- 2004
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 341:3, s. 781-796
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Tidskriftsartikel (refereegranskat)abstract
- Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. P2Ox oxidizes D-glucose and other aldopyranoses regioselectively at C2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at C3. The crystal structure of P2Ox from Trametes multicolor has been determined using single anomalous dispersion with mercury as anomalous scatterer. The model was refined at 1.8 Angstrom resolution to R and R-free values of 0.134 and 0.171, respectively. The overall fold of the P2Ox subunit resembles that of members of the glucose-methanol-choline family of long-chain oxidoreductases, featuring a flavin-binding Rossmann domain of class alpha/beta and a substrate-binding subdomain with a six-stranded central beta sheet and three U helices. The homotetramer buries a large internal cavity of roughly 15,000 Angstrom(3), from which the four active sites are accessible. Four solvent channels lead from the surface into the cavity through which substrate must enter before accessing the active site. The present structure shows an acetate molecule bound in the active site with the carboxylate group positioned immediately below the flavin N5 atom, and with one carboxylate oxygen atom interacting with the catalytic residues His548 and Asn593. The entrance to the active site is blocked by a loop (residues 452 to 461) with excellent electron density but elevated temperature factors. We predict that this loop is dynamic and opens to allow substrate entry and exit. In silico docking of D-glucose in the P2Ox active site shows that with the active-site loop in the closed conformation, monosaccharides cannot be accommodated; however, after removing the loop from the model, a tentative set of protein-substrate interactions for beta-D-glucose have been outlined.
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| 21. |
- Hassan, Noor, et al.
(författare)
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Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis
- 2015
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 99:4, s. 1731-1744
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Tidskriftsartikel (refereegranskat)abstract
- Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial beta-glucosidases (BGL) with significant beta-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesizemore complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific beta-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal beta-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 degrees C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are beta-D-Galp-(1 -> 6)-D-Lac (6GALA) and beta-D-Galp-(1 -> 3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.
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| 22. |
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| 23. |
- Hassan, Noor, 1977-
(författare)
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Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry.In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I).To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS.In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile.Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.
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| 24. |
- Hassan, Noor, et al.
(författare)
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Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly
- 2013
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 3, s. 496-504
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Tidskriftsartikel (refereegranskat)abstract
- The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of β-. d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-. β-. d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17°C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.
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| 25. |
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