A family of starch-active polysaccharide monooxygenases

Abstract

The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.

Keywords: CBM20; copper enzymes; oxygen activation.

Fig. 1.

(A) Representative overall and active site structures of fungal PMOs (PDB ID code 2YET) (10). (B) Structure of cellulose (18, 19). Chitin also contains β(1→4) linkages and has similar crystalline higher order structure to cellulose. (C) Model structure of amylopectin (–25). Hydrogen bonds are shown with green dashed lines.

Fig. 2.

(A) Common domain architecture of 30 predicted starch-active PMOs from different fungal species. Eighteen have the CBM20 domain. (B) Consensus sequence logo representing the putative catalytic domain. Asterisks indicate the absolutely conserved residues also found in cellulose-active PMOs and chitin-active PMOs.

Fig. 3.

Activity assays of NCU08746. Assays contained 5 μM NCU08746 with 2 mM ascorbic acid and atmospheric oxygen. (A and B) Maltodextrins (1–7 units) and soluble portion of amylose (average molecular mass ∼2.8 kDa), respectively, oxidized with Lugol's solution. (C–E) Assays with 50 mg/mL amylopectin, 5 mg/mL PASC, and 50 mg/mL chitin, respectively. The assays were carried out in 50 mM sodium acetate buffer at pH 5.0 and 42 °C.

Fig. 4.

(A) Effect of NCU08746 (5 μM) on the rate of oxidation of MtCDH-2 (1 μM) incubated with 6 μM cellobiose at room temperature. (B) NCU08746 (5 μM) activity assays on amylopectin (50 mg/mL) with 2 mM ascorbic acid (a), with 0.5 μM MtCDH-2 and 5 mM cellobiose (b), and with 0.5 μM MtCDH-2 only (c).

Fig. 5.

k³-weighted EXAFS data of Cu(II)-NCU08746 and its Fourier transform. The boxes highlight the features that arise from the outer-shell atoms of the imidazole ligands. Best fit parameters are provided in SI Appendix, Table S5 (Fit 9).

Fig. 6.

Proposed starch degradation steps by NCU08746 involving the cleavages of α(1→4) (Top and Middle) and α(1→6) (Center and Bottom) linkages via hydroxylation at the C1 position.