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Structural determinants of bacterial lytic polysaccharide monooxygenase functionality.

Zarah Forsberg1, Bastien Bissaro2,3, Jonathan Gullesen2

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Bacterial lytic polysaccharide monooxygenases (LPMOs) regioselectivity in cleaving cellulose and chitin is determined by surface residues near the catalytic copper. Mutations altering substrate interactions impact activity, specificity, and stability.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Bacterial lytic polysaccharide monooxygenases (LPMOs) are enzymes that degrade recalcitrant polysaccharides like cellulose and chitin.
  • LPMO10s utilize redox chemistry involving a copper cofactor to cleave glycosidic bonds.
  • Correlated mutations suggest a network of co-evolved residues on the LPMO surface influences functionality, but their roles are not fully understood.

Purpose of the Study:

  • To investigate the role of co-evolved surface residues in determining the oxidative regioselectivity (C1 vs. C4 oxidation) of LPMO10s.
  • To understand how these residues affect substrate specificity, particularly concerning chitin and cellulose degradation.
  • To explore the impact of mutations on LPMO stability and catalytic efficiency.

Main Methods:

  • Site-directed mutagenesis of specific surface residues in a characterized LPMO10 (MaLPMO10B) from *Micromonospora aurantiaca*.
  • Comparison of mutant enzyme activity against wild-type enzymes with known regioselectivity (C1-oxidizing vs. C1/C4-oxidizing).
  • Assays to determine chitin and cellulose degrading activity, and assessment of enzyme stability under reaction conditions.

Main Results:

  • Mutations in surface residues near the catalytic copper significantly altered the C1:C4 oxidation ratio, with some mutants losing C4 activity while retaining C1 activity.
  • Loss of C4 activity correlated with a loss of chitin-degrading ability, indicating a link between regioselectivity and substrate specificity.
  • Mutations affecting substrate interactions often led to reduced enzyme stability, potentially due to impaired productive substrate binding and increased susceptibility to oxidative self-inactivation.

Conclusions:

  • Surface residues near the catalytic copper are critical determinants of LPMO10 oxidative regioselectivity and substrate specificity.
  • The co-evolution of these residues optimizes multiple LPMO functions, including substrate binding, specificity, catalytic efficiency, and stability.
  • Understanding these structure-function relationships is key to engineering LPMOs for biotechnological applications.