| 1. |
- Abou-Hachem, Maher
(författare)
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Glycoside hydrolases from Rhodothermus marinus Modular organisation and structure-function relationships
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The thermophilic bacterium Rhodothermus marinus produces several thermostable glycoside hydrolases. The studies presented in this thesis were performed on two enzymes, belonging to glycoside hydrolase families 10 and 12, produced by this microorganism. The family 10 xylanase, Xyn10A, is modular in architecture consisting of five domains or modules. The two isolated N-terminal modules were produced and characterised. These modules were proven to be carbohydrate-binding modules (CBMs) belonging to a novel subdivision of family 4 CBMs. Both modules display affinity for xylans, b-glucans, and to a less extent non-crystalline cellulose. The structure of the second of these binding modules (CBM4-2), solved by NMR, featured a b-sandwich with jelly roll-topology. Structural details and substrate titrations provided valuable insight on the determinants of specificity of the module. Both the Xyn10A CBMs and the third domain in the enzyme were shown to bind calcium ions, which had a pronounced effect on their thermostabilities. In addition, modular interactions seemed to enhance the stability of the enzyme, since deletion mutants were less stable than the full-length enzyme. No specific function could be ascribed the third domain of Xyn10A, while evidence suggested that the fifth domain is a novel module type that mediates cell-attachment. The primary structure of the family 12 endoglucanase Cel12A was analysed. These analyses showed that the catalytic module of this enzyme is preceded by a linker sequence and a putative signal peptide that destabilised the enzyme and impaired its expression in Escherichia coli. Designing mutants lacking this signal peptide readily solved the stability and production problems and these mutants retained their thermostability and activity. Finally, fusion proteins between the Xyn10A CBMs and the catalytic module of Cel12A were produced and some of their properties are reported.
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| 2. |
- Abou-Hachem, Maher, et al.
(författare)
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Probing stability of the modular thermostable xylanase Xyn10A
- 2003
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Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1433-4909 .- 1431-0651. ; 7:6, s. 483-491
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Tidskriftsartikel (refereegranskat)abstract
- The thermophilic bacterium Rhodothermus marinus produces a modular xylanase (Xyn10A) consisting of two N-terminal carbohydrate-binding modules (CBMs), followed by a domain of unknown function, and a catalytic module flanked by a fifth domain. Both Xyn10A CBMs bind calcium ions, and this study explores the effect of these ions on the stability of the full-length enzyme. Xyn10A and truncated forms thereof were produced and their thermostabilities were evaluated under different calcium loads. Studies performed using differential scanning calorimetry showed that the unfolding temperature of the Xyn10A was significantly dependent on the presence of Ca2+, and that the third domain of the enzyme binds at least one Ca2+. Thermal inactivation studies confirmed the role of tightly bound Ca2+ in stabilizing the enzyme, but showed that the presence of a large excess of this ion results in reduced kinetic stability. The truncated forms of Xyn10A were less stable than the full-length enzyme, indicative of module/domain thermostabilizing interactions. Finally, possible roles of the two domains of unknown function are discussed in the light of this study. This is the first report on the thermostabilizing role of calcium on a modular family 10 xylanase that displays multiple calcium binding in three of its five domains/modules.
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| 3. |
- Abou-Hachem, Maher, et al.
(författare)
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The modular organisation and stability of a thermostable family 10 xylanase
- 2003
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 21:5-6, s. 253-260
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Tidskriftsartikel (refereegranskat)abstract
- The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca2+ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.
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| 4. |
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| 5. |
- Nordberg Karlsson, Eva, et al.
(författare)
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Rhodothermus marinus: a thermophilic bacterium producing dimeric and hexameric citrate synthase isoenzymes.
- 2002
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Ingår i: Extremophiles. - : Springer Science and Business Media LLC. - 1433-4909 .- 1431-0651. ; 6:1, s. 51-56
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Tidskriftsartikel (refereegranskat)abstract
- Two separate citrate synthases from the extremely thermophilic bacterium Rhodothermus marinus have been identified and purified. One of the enzymes is a hexameric protein and is the first thermostable, hexameric citrate synthase to be isolated. The other is a dimeric enzyme, which is also thermostable but possesses both citrate synthase and 2-methyl citrate synthase activities. 2-Methyl citrate synthase uses propionyl-coenzyme A as one of its substrates and in Escherichia coli, for example, it has been implicated in the metabolism of propionate. However, no growth of R. marinus was observed using minimal medium with propionate as the sole carbon source, and both hexameric and dimeric enzymes were produced irrespective of whether propionate was included in the growth medium. The data are discussed with respect to the evolutionary relationships between the known hexameric and dimeric citrate synthases and 2-methyl citrate synthase.
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| 6. |
- Nordberg Karlsson, Eva, et al.
(författare)
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The modular xylanase Xyn10A from Rhodothermus marinus is cell-attached, and its C-terminal domain has several putative homologues among cell-attached proteins within the phylum Bacteroidetes
- 2004
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 241:2, s. 233-242
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Tidskriftsartikel (refereegranskat)abstract
- Until recently, the function of the fifth domain of the thermostable modular xylanase Xyn10A from Rhodothermus marinus was unresolved. A putative homologue to this domain was however identified in a mannanase (Man26A) from the same microorganism which raised questions regarding a common function. An extensive search of all accessible data-bases as well as the partially sequenced genomes of R. marinus and Cytophaga hutchinsonii showed that homologues of this domain were encoded by multiple genes in microorganisms in the phylum Bacteroidetes. Moreover, the domain occurred invariably at the C-termini of proteins that were predominantly extra-cellular/cell attached. A primary structure motif of three conserved regions including structurally important glycines and a proline was also identified suggesting a conserved 3D fold. This bioinformatic evidence suggested a possible role of this domain in mediating cell attachment. To confirm this theory, R. marinus was grown, and activity assays showed that the major part of the xylanase activity was connected to whole cells. Moreover, immunocytochemical detection using a Xyn10A-specific antibody proved presence of Xyn10A on the R. marinus cell surface. In the light of this, a revision of experimental data present on both Xyn10A and Man26A was performed, and the results all indicate a cell-anchoring role of the domain, suggesting that this domain represents a novel type of module that mediates cell attachment in proteins originating from members of the phylum Bacteroidetes.
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| 7. |
- Teze, David, et al.
(författare)
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The catalytic acid-base in GH109 resides in a conserved GGHGG loop and allows for comparable α-retaining and β-inverting activity in an N-acetylgalactosaminidase from Akkermansia muciniphila
- 2019
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Ingår i: ChemRxiv. - : American Chemical Society (ACS).
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The study describes the first glycoside hydrolase that exhibits comparable levels of activity on α- and β-linked saccharide substrates. This enzyme, assigned into GH109, is encoded by the genome of the human gut symbiont Akkermansia muciniphila that is a model primary degrader of the heavily O-glycosylated mucin glycoprotein that coats the epithelial enterocytes.The elusive catalytic acid/base catalyst in GH109 enzymes is identified as a histidine that is presented by a flexible loop that positions it for catalysis on both α- and β-substrates. This dual activity may be an evolutionary adaptation to extend the range of substrates targeted by a single non-canonical NAD+-dependant GH.
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| 8. |
- Teze, David, et al.
(författare)
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The Catalytic Acid-Base in GH109 Resides in a Conserved GGHGG Loop and Allows for Comparable α-Retaining and β-Inverting Activity in an N-Acetylgalactosaminidase from Akkermansia muciniphila
- 2020
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 10:6, s. 3809-3819
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Tidskriftsartikel (refereegranskat)abstract
- Enzymes active on glycosidic bonds are defined according to the stereochemistry of both substrates and products of the reactions they catalyze. The CAZy classification further assigns these enzymes into sequence-based families sharing a common stereochemistry for substrates (either α- or β-) and products (i.e., inverting or retaining mechanism). Here we describe the N-acetylgalactosaminidases AmGH109A and AmGH109B (i.e., GH109: glycoside hydrolase family 109) from the human gut symbiont Akkermansia muciniphila. Notably, AmGH109A displays α-retaining and β-inverting N-acetylgalactosaminidase activities with comparable efficiencies on natural disaccharides. This dual specificity could provide an advantage in targeting a broader range of host-derived glycans. We rationalize this discovery through bioinformatics, structural, mutational, and computational studies, unveiling a histidine residing in a conserved GGHGG motif as the elusive catalytic acid-base of the GH109 family.
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