Structure of a GH51 α-L-arabinofuranosidase from Meripilus giganteus: Conserved Substrate Recognition from Bacteria to Fungi
Nicholas G.S. McGregor, Johan P. Turkenburg, Kristian B. R. Mørkeberg Krogh, Jens Erik Nielsen, Marta Artola, Keith A. Stubbs, Herman S. Overkleeft and Gideon J. Davies [1]
Molecular Tour
α-L-Arabinofuranosidases play an important role in the degradation of hemicellulosic and pectinaceous plant polysaccharides. Reflecting their importance, arabinofuranosidases are found in several distinct phylogenetic lineages, including glycoside hydrolase (GH) families 43, 51, 54, and 62. Though many α-L-arabinofuranosidase structures have been solved, there remained a significant gap in our understanding of GH51 enzymes; no structure of an industrially relevant fungal GH51 enzyme was known. A lack of sequence similarity between fungal and bacterial enzymes precluded any inference of key function-determining structural features. Of particular interest is the fact that, during the degradation of arabinoxylan, some fungal GH51 enzymes show weak activity towards doubly substituted xylose residues, while others do not. Missing activity towards doubly substituted positions necessitates the inclusion of another enzyme to fully degrade this substrate. Structural understanding of a fungal GH51 α-l-arabinofuranosidase would enable inference of the key structural features of other fungal GH51 enzymes and facilitate their comparison to bacterial homologues.
McGregor et al. describe the first X-ray crystal structure of a fungal GH51 α-L-arabinofuranosidase, MgGH51 from Meripilus giganteus, which was produced industrially in Aspergillus oryzae by Novozymes A/S. The authors were surprisingly unable to solve the phase problem using molecular replacement with existing GH51 models or halide-SAD phasing. Rather than turn to toxic and labour-intensive heavy atom screening, the authors demonstrate the use of a new in vacuo beamline, I23 at Diamond Light source, to rapidly collect high-precision long-wavelength diffraction data and solve the phase problem using native sulphur-SAD signal. This yielded a high-quality model of MgGH51 at 1.2 Å resolution.
Where site-directed mutagenesis has historically been used to study substrate recognition by α-L-arabinofuranosidases from bacteria, McGregor et al. leverage a diverse set of chemical biology tools, including covalent and non-covalent inhibitors, to provide insight into the mechanism and conformational itinerary of the native fungal enzyme. The diverse set of enzyme-ligand complexes presented show how the mechanism, conformational itinerary, and key active site interactions are all conserved between bacterial and fungal GH51 α-L-arabinofuranosidases. However, broader comparison of MgGH51 with TxGH51 reveals significant loop remodelling above and below the active site and the insertion of a domain of unknown function near the N-terminus. Amino acid sequence alignment with other functionally characterised fungal α-L-arabinofuranosidases reveals that these features are conserved among fungal enzymes and points towards key mutations which may give rise to activity towards double substituted xylose residues.
- . The backbone of the polypeptide chain (PDB ID 6zpv) is shown in cartoon coloured blue to red from the N- to C-terminus. The solvent-accessible surface of the enzyme is shown as a transparent white surface. The observed high-mannose N-glycans and a glycerol molecules are shown as sticks with wheat C atoms.
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References
- ↑ McGregor NGS, Turkenburg JP, Morkeberg Krogh KBR, Nielsen JE, Artola M, Stubbs KA, Overkleeft HS, Davies GJ. Structure of a GH51 alpha-L-arabinofuranosidase from Meripilus giganteus: conserved substrate recognition from bacteria to fungi. Acta Crystallogr D Struct Biol. 2020 Nov 1;76(Pt 11):1124-1133. doi:, 10.1107/S205979832001253X. Epub 2020 Oct 16. PMID:33135683 doi:http://dx.doi.org/10.1107/S205979832001253X
