Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

Buchinger, Edith; Knudsen, Daniel H.; Behrens, Manja A.; Pedersen, Jan Skov; Aarstad, Olav Andreas; Tøndervik, Anne; Valla, Svein; Skjåk‐Bræk, Gudmund; Wimmer, Reinhard; Aachmann, Finn Lillelund

doi:10.1074/jbc.m114.567008

Cited by 28 publications

(52 citation statements)

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“…AlyA3 contains an alginate lyase module homologous to AlyA1 and AlyA2 linked to three calcium-binding modules homologous to the R-modules of secreted mannuronan C-5 epimerases (See Mannuronan C-5 Epimerases). It has been shown that R-modules can bind to alginate, although the binding strength is dependent on the specific R-module as well as the structure of the alginate molecule ( Buchinger et al, 2014 ). The cysts of Azotobacter sp.…”

Section: Alginate Lyasesmentioning

confidence: 99%

“…The A-modules are sufficient for both epimerization and for determining the epimerization pattern ( Ertesvåg and Valla, 1999 ). The R-modules are needed for full activity ( Ertesvåg and Valla, 1999 ) and has been shown to participate in substrate binding ( Buchinger et al, 2014 ). At their N-terminal ends each R-module contain four to seven copies of a nine amino acid long motif known to be involved in binding of Ca 2+ ( Baumann et al, 1993 ; Figure 3 ).…”

Section: Mannuronan C-5 Epimerasesmentioning

confidence: 99%

“…The structure of the E4A-module has been determined using x-ray crystallography ( Rozeboom et al, 2008 ), while the structures of the R-modules of AlgE4 and of AlgE6 have been determined using NMR ( Aachmann et al, 2006 ; Buchinger et al, 2014 ). These studies showed that both modules form right-handed β-helixes.…”

Section: Mannuronan C-5 Epimerasesmentioning

confidence: 99%

“…These studies showed that both modules form right-handed β-helixes. A SAXS analysis of full length AlgE4 indicates that it forms a rod-shaped molecule ( Buchinger et al, 2014 ). Both the A- and the R-modules participate in substrate binding, and it has been calculated that the A-module bind 11 uronic acid residues ( Figure 3 ; Tøndervik et al, 2013 ), while the R-module can bind five residues ( Buchinger et al, 2014 ).…”

Section: Mannuronan C-5 Epimerasesmentioning

confidence: 99%

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Alginate-modifying enzymes: biological roles and biotechnological uses

Ertesvåg

2015

Front. Microbiol.

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Alginate denotes a group of industrially important 1-4-linked biopolymers composed of the C-5-epimers β-D-mannuronic acid (M) and α-L-guluronic acid (G). The polysaccharide is manufactured from brown algae where it constitutes the main structural cell wall polymer. The physical properties of a given alginate molecule, e.g., gel-strength, water-binding capacity, viscosity and biocompatibility, are determined by polymer length, the relative amount and distribution of G residues and the acetyl content, all of which are controlled by alginate modifying enzymes. Alginate has also been isolated from some bacteria belonging to the genera Pseudomonas and Azotobacter, and bacterially synthesized alginate may be O-acetylated at O-2 and/or O-3. Initially, alginate is synthesized as polymannuronic acid, and some M residues are subsequently epimerized to G residues. In bacteria a mannuronan C-5-epimerase (AlgG) and an alginate acetylase (AlgX) are integral parts of the protein complex necessary for alginate polymerization and export. All alginate-producing bacteria use periplasmic alginate lyases to remove alginate molecules aberrantly released to the periplasm. Alginate lyases are also produced by organisms that utilize alginate as carbon source. Most alginate-producing organisms encode more than one mannuronan C-5 epimerase, each introducing its specific pattern of G residues. Acetylation protects against further epimerization and from most alginate lyases. An enzyme from Pseudomonas syringae with alginate deacetylase activity has been reported. Functional and structural studies reveal that alginate lyases and epimerases have related enzyme mechanisms and catalytic sites. Alginate lyases are now utilized as tools for alginate characterization. Secreted epimerases have been shown to function well in vitro, and have been engineered further in order to obtain enzymes that can provide alginates with new and desired properties for use in medical and pharmaceutical applications.

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Section: Alginate Lyasesmentioning

confidence: 99%

Section: Mannuronan C-5 Epimerasesmentioning

confidence: 99%

Section: Mannuronan C-5 Epimerasesmentioning

confidence: 99%

Section: Mannuronan C-5 Epimerasesmentioning

confidence: 99%

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Alginate-modifying enzymes: biological roles and biotechnological uses

Ertesvåg

2015

Front. Microbiol.

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“…Several alginate C5 epimerases e.g., ref. 22 , virginiamycin B lyase e.g., ref. 23 and eukaryotic CMLEs 9 have some structural homology to CylK 24 .…”

Section: Discussionmentioning

confidence: 99%

BrtB is an O-alkylating enzyme that generates fatty acid-bartoloside esters

et al. 2020

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Esterification reactions are central to many aspects of industrial and biological chemistry. The formation of carboxyesters typically occurs through nucleophilic attack of an alcohol onto the carboxylate carbon. Under certain conditions employed in organic synthesis, the carboxylate nucleophile can be alkylated to generate esters from alkyl halides, but this reaction has only been observed transiently in enzymatic chemistry. Here, we report a carboxylate alkylating enzyme-BrtBthat catalyzes O-C bond formation between free fatty acids of varying chain length and the secondary alkyl halide moieties found in the bartolosides. Guided by this reactivity, we uncovered a variety of natural fatty acid-bartoloside esters, previously unrecognized products of the bartoloside biosynthetic gene cluster.

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Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum

Fujiwara,

Mano,

Nango

2024

FEBS Letters

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Among the epimerases specific to alginate, some of them in Azotobacter genera convert β‐d‐mannuronic acid to α‐l‐guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C‐5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.

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Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

Cited by 28 publications

References 58 publications

Alginate-modifying enzymes: biological roles and biotechnological uses

Alginate-modifying enzymes: biological roles and biotechnological uses

BrtB is an O-alkylating enzyme that generates fatty acid-bartoloside esters

Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum

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