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, 12 (4), 1095-1103

The Role of the Secondary Coordination Sphere in a Fungal Polysaccharide Monooxygenase

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The Role of the Secondary Coordination Sphere in a Fungal Polysaccharide Monooxygenase

Elise A Span et al. ACS Chem Biol.

Abstract

Polysaccharide monooxygenases (PMOs) are secreted metalloenzymes that catalyze the oxidative degradation of polysaccharides in a copper-, oxygen-, and reductant-dependent manner. Cellulose-active fungal PMOs degrade cellulosic substrates to be utilized as a carbon source for fungal growth. To gain insight into the PMO mechanism, the role of conserved residues in the copper coordination sphere was investigated. Here, we report active-site hydrogen-bonding motifs in the secondary copper coordination sphere of MtPMO3*, a C1-oxidizing PMO from the ascomycete fungus Myceliophthora thermophila. A series of point substitutions that disrupt this conserved network are used to interrogate its function. Activity assays, in conjunction with EPR spectroscopy, demonstrate that residues H161 and Q167 are involved in stabilizing bound oxygen, and H161 appears to play a role in proton transfer. Additionally, Q167 increases the ligand donor strength of Y169 to the copper via a hydrogen-bonding interaction. Altogether, H161 and Q167 are important for oxygen activation, and the results are suggestive of a copper-oxyl active intermediate.

Figures

Figure 1
Figure 1
(a) Crystal structure of MtPMO3* in cartoon representation with primary and secondary coordination sphere residues as sticks. All β-strands are numbered in order of primary sequence. Relevant loop features are highlighted in lavender (L2), pink (LS), teal (L8), and silver (LC). All other carbon atoms are shown in yellow, nitrogen atoms in blue, and oxygen atoms in red. Copper ions are depicted as a brown sphere. (b) MtPMO3* active site showing primary (Nε-Me-H1, H75, Y169) and secondary (T74, H161, Q167) sphere residues with mesh electron density map contoured to 1.5 σ. H161 and Q167 are positioned to H-bond with ligands in the solvent-facing equatorial position. Q167 H-bonds with the axial tyrosine ligand. T74 H-bonds with the N-terminal amino group of H1 and forms a bond with an axial solvent ligand when present. Hydrogen-bonding distances are shown in Ångstroms (Å).
Figure 2
Figure 2
(a) Structural superposition of MtPMO3* (yellow) with three other AA9 structures, PcPMO1 (orange, PDB ID 4B5Q), NcPMO2 (indigo, 4EIR), and NcPMO3 (teal, 4EIS). Conserved residues H161 and Q167 (MtPMO3* numbering) form H-bonds with D26 from a symmetry-related molecule in MtPMO3* (left), a water molecule in NcPMO3 (top right), and a buffer-derived glycerol molecule in PcPMO1 (bottom right). Distances are in Ångstroms (Å). The active-site Cu appears reduced by the X-ray beam in all structures; exogenous molecules depicted in the solvent-facing equatorial position are at lengths (3.6–4.6 Å) from the Cu center that exclude formal coordination. (b) Multiple sequence alignment of representative AA9, AA10, AA11, and AA13 PMOs showing conservation of H-bonding motifs (marked with diamonds) within the PMO superfamily. Fungal PMOs possess a H-X5–8-Q-X-Y (AA9), N-X-E-X-Y (AA11), or Q-X2-Q-X-Y (AA13) motif depending on substrate specificity (cellulose, chitin, and starch, respectively). Bacterial AA10 PMOs active on cellulose contain either a R-X4-E-X-F or H-X2-Q-X-Y motif, correlating with observed regioselectivity (C1 or C1/C4, respectively). Primary coordinating residues are marked with a star. Numbers correspond to MtPMO3* amino acid numbering.
Figure 3
Figure 3
(a) Effect of H-bonding network substitutions on MtPMO3* PASC activity. Assays contained 2 µM PMO, 10 mg mL−1 PASC, atmospheric O2, and 1 µM MtCDH-2 as the reducing agent. Reactions of 45 µL were carried out in 50 mM sodium acetate buffer (pH 5.0) at 40 °C for 1 min. Peaks from aldonic acids with DP 5–13 were quantified via HPAEC with electrochemical detection (nC), and peak areas were integrated over time (nC·min). Smaller C1-oxidized products with DP 2–4 were excluded from this analysis, as they are also products of the CDH reaction (n = 3). (b) Sample HPAEC trace with aldonic acid products labeled by DP, from A2-A13.
Figure 4
Figure 4
Effect of H-bonding network substitutions on O2 consumption rate by MtPMO3*. Assays contained 0.5 µM PMO, 10 mg mL−1 PASC, and atmospheric O2 and were carried out in 50 mM sodium acetate buffer (pH 5.0) at 40 °C. Reactions of 300 µL were initiated with 5 µM MtCDH-2. (a–c) Assay traces showing O2 consumption by variants of H161, Q167, and T74, respectively, in comparison with the wild type (WT) and background (no PMO). (d) Rates of O2 consumption are derived from initial velocity regions after CDH addition (n = 3).
Figure 5
Figure 5
Proposed MtPMO3* reaction mechanisms. Both begin at left with the generally agreed upon Cu(II)–superoxo species that forms upon O2 binding to MtPMO3*-Cu(I). The top half follows one possible mechanism based on hydrogen atom transfer (HAT) by the superoxo intermediate, followed by reduction and cleavage of the distal O atom of the hydroperoxo intermediate to form water and an oxyl intermediate that undergoes radical rebound with the substrate. After substrate release, a one-electron reduction returns the Cu(I) enzyme (at right). The bottom half follows a possible mechanism utilizing an oxyl species for HAT, which is formed by reducing and cleaving the terminal O atom of the O2 adduct to form water. Uncoupling of oxygen activation from substrate hydroxylation could follow the middle pathway that produces peroxide. The release of superoxide from the H161 variants shows that H161 plays a proton transfer role to the Cu(II)-superoxo species, at least in the uncoupled reaction.
Figure 6
Figure 6
X-band EPR spectra (black) of wild type MtPMO3* and six H-bonding network variants, with simulations (red) for WT and the Q167A variant. Spectra were recorded at 40 K, 9.4 GHz, 0.2 mW power, and 0.5 mT modulation amplitude and have been scaled for signal strength. The signal at ~335 mT in the WT spectra is a feature from the cavity. Simulation parameters for WT MtPMO3*: g = [2.260 2.079 2.032], A(63/65Cu) = [504 30 25] MHz, 3x A(14N) = [40 40 40] MHz, g-strain = [0.007 0.007 0.007]. Simulation parameters for the Q167A variant: g = [2.246 2.055 2.049], A(63/65Cu) = [558 56 20] MHz, 3x A(14N) = [37.6 37.6 37.6] MHz, g-strain = [0.007 0.007 0.007].
Figure 7
Figure 7
(A) H-bonding to Y169 in wild type (WT) MtPMO3* and the Q167E variant. (B) Geometric and electronic effects of removing a H-bond acceptor to Y169, as in the Q167A variant: elongation of the Cu–Tyr distance and contraction of the Cu–equatorial ligand distances results in an increase in the energy between d(x2y2) and d(xy).
Scheme 1
Scheme 1. Polysaccharide Monooxygenase (PMO) Reaction, R–H = Substrate (e.g., Cellulose)

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