The molecular basis of endolytic activity of a multidomain alginate lyase from Defluviitalea phaphyphila, a representative of a new lyase family, PL39

Ji, Shaocheng; Dix, S.R.; Aziz, Adli A; Sedelnikova, Svetlana E.; Baker, Patrick J.; Rafferty, John B.; Bullough, Per A.; Tzokov, Svetomir B.; Agirre, Jon; Li, Fuli; Rice, David W.

doi:10.1074/jbc.ra119.010716

Cited by 42 publications

(38 citation statements)

References 58 publications

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“…Work is currently being done on the Sails program to be able to overcome many of these limitations. In addition, based on encouraging early results [63][64][65][66], new carbohydrate dictionaries with more faithful model geometry and accurate torsion restraints will improve refinement, particularly for cryo-EM. Finally, sugars in active sites of enzymes might be distorted into high energy conformations, and thus may require further validation; work will need to be done in this respect in order to give users a confidence level on their conformational assignment.…”

Section: Future Perspectivesmentioning

confidence: 99%

Structural glycobiology in the age of electron cryo-microscopy

Atanasova

Bagdonas

Agirre

2020

Current Opinion in Structural Biology

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Highlights • Progress is being made on providing automated model building and validation tools for structural glycobiology • Electron cryo-microscopy (cryo-EM) can now be routinely used for resolving protein glycosylation • High-resolution cryo-EM structures show fewer pyranose high-energy conformations than X-ray ones • Re-refinement with the latest methods can produce better structures of glycoproteins automatically

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Section: Future Perspectivesmentioning

confidence: 99%

Structural glycobiology in the age of electron cryo-microscopy

Atanasova

Bagdonas

Agirre

2020

Current Opinion in Structural Biology

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“…1 ). The N-terminal catalytic domain consists of a five-bladed β-propeller (Gln 21 –Gly 325 ), as in other GH clan-F enzymes, and the C-terminal domain ( Pc CBM35) takes a β-jellyroll fold (Thr 326 –Tyr 448 ) structure, as in previously reported CBM35s ( 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ). Pc CBM35 contains one calcium ion near the end of the first β-strand on a different domain surface from the plane to which the ligand binds ( Fig.…”

Section: Resultsmentioning

confidence: 73%

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-β-1,3-galactanase from Phanerochaete chrysosporium

Matsuyama

Kishine

Fujimoto

et al. 2020

Journal of Biological Chemistry

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Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family (CBM) 35 binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveal that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly->Trp substitution, which affects pyranose stacking, and an Asp->Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.

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“…1). The N-terminal catalytic domain consists of a five-bladed b-propeller (Gln21-Gly325), as in other GH clan-F enzymes, and the C-terminal domain (PcCBM35) takes a β-jellyroll fold (Thr326-Tyr448) structure, as in previously reported CBM35s (16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

Section: Overall Structure Of Pc13gal43amentioning

confidence: 80%

Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycetePhanerochaete chrysosporiumprovides insights into the mechanism of bypassing side chains

Matsuyama

Kishine

Fujimoto

et al. 2020

Preprint

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Arabinogalactan proteins (AGPs) are functional plant proteoglycans, but their functions are largely unexplored, mainly because of the complexity of the sugar moieties, which are generally analyzed with the aid of glycoside hydrolases. In this study, we solved the apo and liganded structures of exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. It is composed of a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family (CBM) 35 binding domain. GH43_sub24 lacks the catalytic base Asp that is conserved among other GH43 subfamilies. Crystal structure and kinetic analyses indicated that the tautomerized imidic acid function of Gln263 serves instead as the catalytic base residue. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s. Some of the residues involved in ligand recognition differ from those of galactomannan-binding CBM35, including substitution of Trp for Gly, which affects pyranose stacking, and substitution of Asn for Asp in the lower part of the binding pocket. Pc1,3Gal43A WT and its mutants at residues involved in substrate recognition are expected to be useful tools for structural analysis of AGPs. Our findings should also be helpful in engineering designer enzymes for efficient utilization of various types of biomass.

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The molecular basis of endolytic activity of a multidomain alginate lyase from Defluviitalea phaphyphila, a representative of a new lyase family, PL39

Cited by 42 publications

References 58 publications

Structural glycobiology in the age of electron cryo-microscopy

Structural glycobiology in the age of electron cryo-microscopy

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-β-1,3-galactanase from Phanerochaete chrysosporium

Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycetePhanerochaete chrysosporiumprovides insights into the mechanism of bypassing side chains

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