Journal:Acta Cryst D:S205979832001253X
From Proteopedia
Structure of a GH51 α-L-arabinofuranosidase from Meripilus giganteus: Conserved Substrate Recognition from Bacteria to FungiNicholas 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 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. Overview of the tertiary structure of MgGH51. 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. Overlay of the AraDNJ and α-L-AraAZI ligands within the MgGH51 active site. The active site residues and ligand from the α-L-AraAZI complex (6zq0) are shown as grey ball-and-sticks. The active site residues and ligand from the AraDNJ complex (6zq1) are shown as cyan ball-and-sticks. Apparent hydrogen bonding interactions are shown as white dashes. A simple sequence alignment between MgGH51 and T. xylanilyticus GH51 (TxGH51; PDB entry 2vrq), a structurally characterized bacterial homologue, finds only 23% sequence identity. However, structural superposition of MgGH51 onto TxGH51 using only arabinofuranose, the general acid/base and the catalytic nucleophile reveals significant overall structural homology (MgGH51 is colored in magenta and TxGH51 in tan). The active sites of the two enzymes are nearly identical, with absolute conservation of Tyr402 (Tyr242 in TxGH51), Glu23 (Glu28 in TxGH51), Asn350 (Asn175 in TxGH51), the Asn231 backbone (the Cys74 backbone in TxGH51), Glu351 (Glu176 in TxGH51) and Glu429 (Glu298 in TxGH51). The only polar contact that is not conserved between the two active sites is the interaction between Gln347 and O5 found only in TxGH51. PDB references: MgGH51, unliganded, crystal type 1, 6zpy; crystal type 2, 6zpx; crystal type 3, 6zpv; crystal type 3, collected at 2.75 Å wavelength, 6zps; complex with α-L-AraCS, crystal type 1, 6zpw; complex with arabinose, crystal type 1, 6zpz; complex with α-L-AraAZI, crystal type 1, 6zq0; complex with AraDNJ, crystal type 1, 6zq1. References
|
| ||||||||||
