SHORTCUTS TO MUSHROOMS: NMR AND MS ANALYSES
OF FUNGAL PYRANOSE OXIDASE
P. Halada1, P. Sedmera1, C. Leitner2, K. Bezouška1, D. Haltrich2 and J. Volc1
1Institute of Microbiology, Academy of Sciences of the Czech Republic,
Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic
2Division of Biochemical Engineering, Institute of Food Technology,
University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria
Pyranose 2-oxidase (P2O, EC 184.108.40.206), a fungal periplasmic homotetrameric flavoprotein (~300 kDa), has received increased attention due to its potential analytical and biotechnological applications . This enzyme catalyzes C-2/C-3 oxidation of numerous sugars to their corresponding dicarbonyl derivatives (aldos-2-uloses or glycosid-3-uloses), accompanied with the reduction of flavin adenine dinucleotide (FAD), an obligatory cofactor. P2O has a great biotechnological potential as a catalyst in the key step of C-2 oxidation of D-glucose and D-galactose in the production of modern low-caloric sweeteners D-fructose and D-tagatose.
Our research on the P2O from the fungus Trametes multicolor followed three subjects. The first one was the study of the enzyme substrate specificity and characterization of its reaction products by spectral analyses (NMR, FAB mass spectrometry) [2,3]. Further, we applied MALDI mass spectrometry with post-source decay (PSD) analysis to determine sequence segments suitable for designing PCR primers for cloning cDNA corresponding to the P2O gene . Finally, we elucidated the structure of the P2O flavin-binding domain, which is of importance for understanding the enzyme reaction mechanisms and possible optimized application. The combination of PSD-MALDI MS and electrospray ion trap mass spectrometry (ESI IT-MS) on the isolated flavopeptide identified flavopeptide sequence, flavin type and flavin linkage site. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy resulting in the structure STXW with X = 8α(N 3histidyl)-FAD .
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3. J. Volc, P. Sedmera, P. Halada, P. Dwivedi & M. Costa-Ferreira, J. Carb. Chem., in press (2003).
4. P. Halada, C. Leitner, J. Volc, D. Haltrich & V. Havlíček, Collect. Czech. Chem. Commun. 65 (2000) 1669-1676.
5. P. Halada, C. Leitner, P. Sedmera, D. Haltrich & J. Volc, Anal. Biochem., in press, (2003).
The work was supported by Program for Scientific-Technical Cooperation AKTION, Austria-Czech Republic (2002–9) and Institutional Research Concept AV0Z5020903.