title: Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly.
abstract: The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of β-d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-β-d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17 °C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.
authors.affiliation: KTH Royal Institute of Technology, School of Biotechnology, Albanova University Center, Roslagstullsbacken 21, S-10691 Stockholm Sweden.
authors.forename: Noor
authors.initials: N
authors.lastname: Hassan
authors.forename: Tien-Chye
authors.initials: TC
authors.lastname: Tan
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
authors.forename: Ines
authors.initials: I
authors.lastname: Pisanelli
authors.forename: Laura
authors.initials: L
authors.lastname: Fusco
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
authors.forename: Christina
authors.initials: C
authors.lastname: Divne
keywords: 2FGlc, 2-deoxy-2-fluoro-d-glucose; 3FGlc, 3-deoxy-3-fluoro-d-glucose; Crystal structure; DTT, dithiothreitol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IMAC, by immobilized metal ion affinity chromatography; IPTG, β-d-1-thiogalactopyranoside; Lignin degradation; MES, 2-(N-morpholino) ethanesulfonic acid; MWCO, molecular weight cut off; Oligomerization; P2O, pyranose oxidase; PBS, phosphate buffered saline; PDB, Protein Data Bank; PEG, polyethylene glycol; Propeptide; Pyranose 2-oxidase; TEV, Tobacco Etch Virus; Thermostability