A quantitative indicator diagram for lytic polysaccharide monooxygenases reveals the role of aromatic surface residues in HjLPMO9A regioselectivity

Lytic polysaccharide monooxygenases ( LPMOs) have changed our understanding of lignocellulosic degradation dramatically over the last years. These metalloproteins catalyze oxidative cleavage of recalcitrant polysaccharides and can act on the C1 and/or C4 position of glycosidic bonds. Structural data have led to several hypotheses, but we are still a long way from reaching complete understanding of the factors that determine their divergent regioselectivity. Site-directed mutagenesis enables the investigation of structure-function relationship in enzymes and will be of major importance in unraveling this intriguing matter. In this context, it is crucial to have an enzyme assay or screening approach with a direct correlation with the desired functionality. LPMOs render this search extra challenging due to their insoluble substrates, complex pattern of reaction products and lack of synthetic standards of most oxidized products. Here, we describe a regioselectivity indicator diagram based on the time-course of only 2 HPAEC-PAD signals. The diagram was successfully used to confirm the hypothesis that aromatic surface residues influence the C1/C4 oxidation ratio in Hypocrea jecorina LPMO9A. Consequently, the diagram should become a valuable tool in the search towards better understanding and engineering of regioselectivity in LPMOs

A novel cytosolic NADH:quinone oxidoreductase from Methanothermobacter marburgensis

Methanothermobacter marburgensis is a strictly anaerobic, thermophilic methanogenic archaeon that uses methanogenesis to convert H2 and CO2 to energy. M. marburgensis is one of the best-studied methanogens, and all genes required for methanogenic metabolism have been identified. Nonetheless, the present study describes a gene (Gene ID 9704440) coding for a putative NAD(P)H:quinone oxidoreductase that has not yet been identified as part of the metabolic machinery. The gene product, MmNQO, was successfully expressed, purified and characterized biochemically, as well as structurally. MmNQO was identified as a flavin-dependent NADH:quinone oxidoreductase with the capacity to oxidize NADH in the presence of a wide range of electron acceptors, whereas NADPH was oxidized with only three acceptors. The 1.50 Å crystal structure of MmNQO features a homodimeric enzyme where each monomer comprises 196 residues folding into flavodoxin-like α/β domains with non-covalently bound FMN (flavin mononucleotide). The closest structural homologue is the modulator of drug activity B from Streptococcus mutans with 1.6 Å root-mean-square deviation on 161 Cα atoms and 28% amino-acid sequence identity. The low similarity at sequence and structural level suggests that MmNQO is unique among NADH:quinone oxidoreductases characterized to date. Based on preliminary bioreactor experiments, MmNQO could provide a useful tool to prevent overflow metabolism in applications that require cells with high energy demand

HPAEC-PAD chromatograms of all 3 LPMO representatives.

<p>Activity of <i>Hj</i>LPMO9A (red), <i>Nc</i>LPMO9C (green), <i>Pc</i>LPMO9D (blue) and wildtype <i>P</i>. <i>pastoris</i> CBS7435 broth (black) on 0.5% PASC in the presence of 1 mM ascorbic acid as reducing agent. The six signals (A1-3 and K1-3) that will be evaluated for their use as indicator signal are indicated in the top chromatogram.</p

LPMO regioselectivity.

<p>Oxidation of the C1 position generates a lactone, which is hydrated to a reducing-end aldonic acid. C4-oxidation leads to non-reducing-end 4-ketoaldose formation, which will spontaneously hydrate to gemdiols in aqueous conditions.</p

Indicator diagram applied to the three LPMO regioselectivity types.

<p>Preliminary evaluation of the indicator diagram was done by incubating a member of each LPMO regioselectivity type on PASC: ● = <i>Hj</i>LPMO9A (C1/C4-oxidizer, 1.2–12 μM), ▼ = <i>Pc</i>LPMO9D (C1-oxidizer, 1–10 μM), ○ = <i>Nc</i>LPMO9C (C4-oxidizer, 0.9–2.8 μM).</p

Indicator diagram demonstrates role of aromatic residues in LPMO regioselectivity.

<p>Slopes, which are a measure of the ratio of C4/C1-oxidation, are listed next to the regression line, together with their standard deviation. ● = <i>Hj</i>LPMO9A wildtype (1.4–14 μM), ○ = Y24A variant (1.2–12 μM), ▼ = Y211 (1.2–12 μM) variant.</p

Aromatic surface residues in the C1/C4-oxidizing <i>Hj</i>LPMO9A.

<p>(A) Homology model of <i>Hj</i>LPMO9A (based on 3ZUD as template) with the aromatic surface residues selected for alanine scanning in pink stick representation. Active site residues are shown as yellow sticks, the copper ion as a blue sphere. (B) 3DM structure based multiple sequence alignment [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178446#pone.0178446.ref040" target="_blank">40</a>] of AA9 characterized LPMOs with known regioselectivity. The residues aligned with the Y24, F43, W84 and Y211 aromatic surface residue of <i>Hj</i>LPMO9A are highlighted in pink. Residues in the 3DM core alignment are represented by capitals, the alignment of structurally variable regions are in lower case. The insertion typical for most C1/C4-oxidizing LPMOs is marked in yellow.</p

Correlation between release speeds of aldonic acid / 4-ketoaldose peaks and the <i>Hj</i>LPMO9A LPMO load in 500 μL reaction mixture.

<p>Graph A represents the aldonic acid peaks (● = A1, ○ = A2, ▼ = A3); Graph B represents the 4-ketoaldose peaks (■ = K1, □ = K2, ▲ = K3).</p

Effect of mutating aromatic surface residues on <i>Hj</i>LPMO9A regioselectivity.

<p>This effect is determined by comparing the C1/C4-oxidation ratio (slope in the indicator diagram) of the wildtype enzyme (1.4–14 μM) and the variants (1.2–12 μM).</p