Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus

Emil G. P. Stender, Christian Dybdahl Andersen, Folmer Fredslund, Jesper Holck, Amalie Solberg, David Teze, Günther H.J. Peters, Bjørn E. Christensen, Finn L. Aachmann, Ditte Hededam Welner, Birte Svensson*

*Corresponding author for this work

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Abstract

Alginate is a linear polysaccharide from brown algae consisting of 1,4 linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% of that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and tri-saccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC-electrospray ionization (ESI)-MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.
Original languageEnglish
Article numberjbc.RA119.010206
JournalJournal of Biological Chemistry
Volume294
Issue number47
Pages (from-to)17915-17930
Number of pages17
ISSN0021-9258
DOIs
Publication statusPublished - 2019

Keywords

  • Alginate lyase
  • Bacteroides cellulosilyticus CRE21
  • Crystal structure
  • Parallel β-helix
  • Asparagine ladder
  • Molecular docking
  • Enzyme kinetics
  • Enzyme mechanism
  • Mutational analysis
  • Imidazole rescue

Cite this

@article{b95b306fd67c473ba2a33dbd60035530,
title = "Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus",
abstract = "Alginate is a linear polysaccharide from brown algae consisting of 1,4 linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 {\AA} resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5{\%} of that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and tri-saccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC-electrospray ionization (ESI)-MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.",
keywords = "Alginate lyase, Bacteroides cellulosilyticus CRE21, Crystal structure, Parallel β-helix, Asparagine ladder, Molecular docking, Enzyme kinetics, Enzyme mechanism, Mutational analysis, Imidazole rescue",
author = "Stender, {Emil G. P.} and Andersen, {Christian Dybdahl} and Folmer Fredslund and Jesper Holck and Amalie Solberg and David Teze and Peters, {G{\"u}nther H.J.} and Christensen, {Bj{\o}rn E.} and Aachmann, {Finn L.} and Welner, {Ditte Hededam} and Birte Svensson",
year = "2019",
doi = "10.1074/jbc.RA119.010206",
language = "English",
volume = "294",
pages = "17915--17930",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "47",

}

Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus. / Stender, Emil G. P.; Andersen, Christian Dybdahl; Fredslund, Folmer; Holck, Jesper; Solberg, Amalie; Teze, David; Peters, Günther H.J.; Christensen, Bjørn E.; Aachmann, Finn L.; Welner, Ditte Hededam; Svensson, Birte.

In: Journal of Biological Chemistry, Vol. 294, No. 47, jbc.RA119.010206, 2019, p. 17915-17930.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus

AU - Stender, Emil G. P.

AU - Andersen, Christian Dybdahl

AU - Fredslund, Folmer

AU - Holck, Jesper

AU - Solberg, Amalie

AU - Teze, David

AU - Peters, Günther H.J.

AU - Christensen, Bjørn E.

AU - Aachmann, Finn L.

AU - Welner, Ditte Hededam

AU - Svensson, Birte

PY - 2019

Y1 - 2019

N2 - Alginate is a linear polysaccharide from brown algae consisting of 1,4 linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% of that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and tri-saccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC-electrospray ionization (ESI)-MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.

AB - Alginate is a linear polysaccharide from brown algae consisting of 1,4 linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% of that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and tri-saccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC-electrospray ionization (ESI)-MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.

KW - Alginate lyase

KW - Bacteroides cellulosilyticus CRE21

KW - Crystal structure

KW - Parallel β-helix

KW - Asparagine ladder

KW - Molecular docking

KW - Enzyme kinetics

KW - Enzyme mechanism

KW - Mutational analysis

KW - Imidazole rescue

U2 - 10.1074/jbc.RA119.010206

DO - 10.1074/jbc.RA119.010206

M3 - Journal article

VL - 294

SP - 17915

EP - 17930

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 47

M1 - jbc.RA119.010206

ER -