Second-Sphere Histidine Catalytic Function in a Fungal Polysaccharide Monooxygenase

Area of Science:

• Biochemistry
• Enzymology
• Carbohydrate Chemistry

Background:

• Fungal polysaccharide monooxygenases (PMOs) are copper enzymes that degrade carbohydrates.
• Auxiliary activity 9 (AA9) family PMOs possess a conserved histidine residue in their second-sphere region.
• This histidine is hypothesized to be involved in proton transfer during O2-dependent reactions.

Purpose of the Study:

• To investigate the role of the second-sphere histidine (H157) in the AA9 enzyme MtPMO9E.
• To determine if H157 is crucial for proton transfer or oxygen intermediate stabilization in PMO catalysis.

Main Methods:

• Site-directed mutagenesis was used to create H157A and H157Q variants of MtPMO9E.
• Enzyme kinetics were performed using cellohexaose (Glc6) as a substrate.
• Oxidase and hydroxylation activities were measured with O2 and H2O2 as cosubstrates.
• Crystal structures of WT and H157Q MtPMO9E were compared.

Main Results:

• H157A and H157Q variants showed similar folding and oxidase activity to wild-type (WT) MtPMO9E.
• Variants exhibited reduced affinity for Glc6 and lower hydroxylation activity compared to WT.
• H157A showed the least activity, while H157Q retained some activity with H2O2.
• Structural analysis revealed Q157 in the variant overlays with H157 in WT.

Conclusions:

• The second-sphere histidine (H157) is not essential for proton transfer in MtPMO9E.
• H157 functions as a hydrogen-bond donor to diatomic oxygen intermediates.
• This interaction facilitates catalysis by both O2 and H2O2 in fungal PMOs.