Back to Search Start Over

Second-Sphere Histidine Catalytic Function in a Fungal Polysaccharide Monooxygenase.

Second-Sphere Histidine Catalytic Function in a Fungal Polysaccharide Monooxygenase.

Authors :
Batka AE
Thomas WC
Tudorica DA
Sayler RI
Marletta MA
Source :
Biochemistry [Biochemistry] 2024 Dec 03; Vol. 63 (23), pp. 3136-3146. Date of Electronic Publication: 2024 Nov 20.
Publication Year :
2024

Abstract

Fungal polysaccharide monooxygenases (PMOs) oxidatively degrade cellulose and other carbohydrate polymers via a mononuclear copper active site using either O <subscript>2</subscript> or H <subscript>2</subscript> O <subscript>2</subscript> as a cosubstrate. Cellulose-active fungal PMOs in the auxiliary activity 9 (AA9) family have a conserved second-sphere hydrogen-bonding network consisting of histidine, glutamine, and tyrosine residues. The second-sphere histidine has been hypothesized to play a role in proton transfer in the O <subscript>2</subscript> -dependent PMO reaction. Here the role of the second-sphere histidine (H157) in an AA9 PMO, Mt PMO9E, was investigated. This PMO is active on soluble cello-oligosaccharides such as cellohexaose (Glc6), thus enabling kinetic analysis with the point variants H157A and H157Q. The variants appeared to fold similarly to the wild-type (WT) enzyme and yet exhibited weaker affinity toward Glc6 than WT (WT K <subscript>D</subscript> = 20 ± 3 μM). The variants had comparable oxidase (O <subscript>2</subscript> reduction to H <subscript>2</subscript> O <subscript>2</subscript> ) activity to WT at all pH values tested. Using O <subscript>2</subscript> as a cosubstrate, the variants were less active for Glc6 hydroxylation than WT, with H157A being the least active. Similarly, H157Q was competent for Glc6 hydroxylation with H <subscript>2</subscript> O <subscript>2</subscript> , but H157A was less active. Comparison of the crystal structures of H157Q and WT Mt PMO9E reveals that a terminal heteroatom of Q157 overlays with N <subscript>ε</subscript> of H157. Altogether, the data suggest that H157 is not important for proton transfer, but support a role for H157 as a hydrogen-bond donor to diatomic-oxygen intermediates, thus facilitating catalysis with either O <subscript>2</subscript> or H <subscript>2</subscript> O <subscript>2</subscript> .

Details

Language :
English
ISSN :
1520-4995
Volume :
63
Issue :
23
Database :
MEDLINE
Journal :
Biochemistry
Publication Type :
Academic Journal
Accession number :
39563485
Full Text :
https://doi.org/10.1021/acs.biochem.4c00527