Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover

Petr Halada,Clemens K Peterbauer,Christian Leitner,Dagmar Brugger,Jindrich Volc,Dietmar Haltrich,Daniel Cullen

doi:10.1371/journal.pone.0148108

Abstract

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.

Highlights

Pyranose oxidase is a ~270 kDa, 8α-(N3)-histidyl flavinylated, homotetrameric flavin-dependent oxidase
Plasmid DNA was transformed into electrocompetent E. coli BL21 Star DE3 cells and after regeneration the cells were grown over night at 37°C on LBamp plates (1% peptone, 0.5% yeast extract, 1% NaCl, 1.4% agar supplemented with 100 mg mL-1 ampicillin)
When pyranose oxidase from T. multicolor (TmPOx) was incubated with 100 mM D-glucose under aerobic conditions and in the absence of catalase, approximately half of the activity was lost within 90 min, and after 4 h of incubation only 6% of the initial activity was retained

Summary

Introduction

Pyranose oxidase (other names are glucose 2-oxidase or pyranose 2-oxidase, POx; systematic name pyranose:oxygen 2-oxidoreductase; EC 1.1.3.10) is a ~270 kDa, 8α-(N3)-histidyl flavinylated, homotetrameric flavin-dependent oxidase. Plots of residual activity versus time (Fig 1) showed that POx was inactivated by H2O2 in a time- and concentration-depending process following first-order kinetics.

Results

Conclusion