Cello-oligosaccharide oxidation reveals differences between two lytic polysaccharide monooxygenases (family GH61) from Podospora anserina

Abstract

The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

Fig 1

SDS-PAGE (lanes 1 to 3) and zymogram analysis (lanes 4 to 7) of PaGH61 enzymes. For SDS-PAGE, lane 1, prestained molecular mass marker; lane 2, 10 μg of purified PaGH61A; lane 3, 10 μg of purified PaGH61B; for a CMC zymogram under native conditions incubated overnight at 45°C under shaking with addition of 10 mM ascorbic acid, lane 4, 50 μg of rPaGH61B; lane 5, 50 μg of PaGH61A; for a CMC zymogram under denaturing conditions, lane 6, 150 μg of purified PaGH61B; lane 7, 150 μg of purified PaGH61A.

Fig 2

Products generated following the synergistic action of PaGH61 and CDH on cellulose. Analysis was performed using HPAEC-PAD after 48 h of incubation at 45°C under shaking of 1% (wt/vol) PASC in 50 mM sodium acetate (pH 5) with CDH/PaGH61. (A, B) Action of PaGH61A at 5 mg · g⁻¹ (A) and PaGH61B at 5 mg · g⁻¹ (B) in association with 500 μg · g⁻¹ of CDH; (C) products formed by combination of PaGH61B (50 mg · g⁻¹) with 500 μg · g⁻¹ of CDH. All experiments were carried out in triplicate. nC, nanocoulomb.

Fig 3

MALDI TOF/TOF spectra of products following the combined action of PaGH61 with CDH. Analysis was performed after 48 h of incubation at 45°C under shaking of 1% (wt/vol) PASC in 50 mM sodium acetate (pH 5). (A) PaGH61A (5 mg · g⁻¹) combined with CDH (500 μg · g⁻¹); (B) PaGH61B (5 mg · g⁻¹) combined with CDH (500 μg · g⁻¹); (C) PaGH61B (50 mg.g⁻¹) combined with CDH (500 μg · g⁻¹). MS/MS fragmentation was performed on products released at m/z 1,045 (D) and m/z 1,245 (E) under the same conditions described for panel C. Identified compounds are labeled in black for native cellodextrins and in red when the products are oxidized. aa, aldonic acid; al, aldonolactone/lactone; OI, oxygen incorporation; *, compounds are subject to hypothesis. The numbers in different-colored boxes refer to the masses of compounds identified during MS/MS fragmentation.

Fig 3

MALDI TOF/TOF spectra of products following the combined action of PaGH61 with CDH. Analysis was performed after 48 h of incubation at 45°C under shaking of 1% (wt/vol) PASC in 50 mM sodium acetate (pH 5). (A) PaGH61A (5 mg · g⁻¹) combined with CDH (500 μg · g⁻¹); (B) PaGH61B (5 mg · g⁻¹) combined with CDH (500 μg · g⁻¹); (C) PaGH61B (50 mg.g⁻¹) combined with CDH (500 μg · g⁻¹). MS/MS fragmentation was performed on products released at m/z 1,045 (D) and m/z 1,245 (E) under the same conditions described for panel C. Identified compounds are labeled in black for native cellodextrins and in red when the products are oxidized. aa, aldonic acid; al, aldonolactone/lactone; OI, oxygen incorporation; *, compounds are subject to hypothesis. The numbers in different-colored boxes refer to the masses of compounds identified during MS/MS fragmentation.

Fig 4

Schematic representation of products formed by the synergistic action of PaGH61 and CDH.