title: Further insights into the catalytical properties of deglycosylated pyranose dehydrogenase from Agaricus meleagris recombinantly expressed in Pichia pastoris.
abstract: The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris . Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 °C. The remaining larger fragment has a molecular weight of ∼46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, "wired" to graphite electrodes with two different osmium redox polymers: [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly-(vinylimidazole))10Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with Jmax 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N(75), N(175), and N(252) were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University , PO Box 124, 221 00 Lund, Sweden.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Yakovleva
authors.forename: Anikó
authors.initials: A
authors.lastname: Killyéni
authors.forename: Oliver
authors.initials: O
authors.lastname: Seubert
authors.forename: Peter
authors.initials: P
authors.lastname: O Conghaile
authors.forename: Domhnall
authors.initials: D
authors.lastname: Macaodha
authors.forename: Dónal
authors.initials: D
authors.lastname: Leech
authors.forename: Christoph
authors.initials: C
authors.lastname: Gonaus
authors.forename: Ionel Catalin
authors.initials: IC
authors.lastname: Popescu
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
authors.forename: Sven
authors.initials: S
authors.lastname: Kjellström
authors.forename: Lo
authors.initials: L
authors.lastname: Gorton
mesh_terms: D000364:Agaricus / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D055162:Biocatalysis; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004563:Electrochemistry; D015870:Gene Expression; D006031:Glycosylation; D009005:Monosaccharides / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D009005:Monosaccharides; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases
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