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PRINCIPAL PUBLICATION AND AUTHORS

AA16 Oxidoreductases Boost Cellulose-Active AA9 Lytic Polysaccharide Monooxygenases from Myceliophthora thermophila, P. Sun (a), Z. Huang (b), S. Banerjee (b), M.A.S. Kadowaki (c), R.J. Veersma (a), S. Magri (c), R. Hilgers (a), S.J. Muderspach (b), C.V.F.P. Laurent (d), R. Ludwig (d), D. Cannella (c), L. Lo Leggio (b), W.J.H. van Berkel (a), M.A. Kabel (a), ACS Catal. 13, 7, 4454-4467 (2023); https:/doi.org/10.1021/acscatal.3c00874 (a) Wageningen University, Wageningen (The Netherlands) (b) University of Copenhagen, Copenhagen (Denmark) (c) Université Libre de Bruxelles, Brussels (Belgium) (d) University of Natural Resources and Life Sciences (BOKU), Vienna (Austria)

REFERENCES

[1] B.L Cantarel et al., Nucleic Acids Res. 37, D233-D238 (2009). [2] T. Tandrup et al., Biochem. Soc. Trans. 46, 1431-1447 (2018). [3] S. Kuusk et al., J. Biol. Chem. 293, 523-531 (2018). [4] C. Filiatrault-Chastel et al., Biotechnol. Biofuels 12, 55 (2019).

compounds like syringol in the laboratory, though this is unlikely to represent the biological function of MtAA16A (Figure 89). MtAA16A thus appears to be one of the many new LPMO-like proteins, with strong structural similarity to LPMOs, but having evolved a different function.

In search of a different function, further biochemical characterisation indicated that MtAA16A had significant oxidase activity (H2O2 production), which could boost the catalytic function of true LPMOs. While boosting LPMOs through in-situ H2O2 production, including enzymatic production, has previously been shown, this boosting was remarkably specific for LPMOs from Myceliophthora thermophila (MtAA9s), while other tested LPMOs from Neurospora crassa (NcAA9s) were not boosted. One explanation for this unprecedented boosting specificity could be the specific formation of transient protein-protein complexes, where the formed H2O2 by MtAA16A might easily reach the catalytic site of MtLPMO9s to boost the degradation of cellulose. This would provide an advantage as the overall concentrations of H2O2 can remain low, limiting oxidative damage to proteins. Plausible structural models for such transient interactions await experimental validation.

With a biorefinery perspective, the overall conclusion of this work is that histidine brace enzymes can have additional beneficial functions other than direct cleavage of recalcitrant polysaccharides in this case, in-situ production and delivery of H2O2 specifically where needed. Further research in the interactions between such enzymes has perspectives in reducing overall enzyme load, thereby reducing cost and CO2 footprint in processing lignocellulosic waste for sustainable applications.

Fig. 89: Left: Close-up structure of the active site of MtAA16A with electron density. Right: Small surface pocket near the MtAA16A active site.