Enzymatic versatility and thermostability of a new aryl-alcohol oxidase from Thermothelomyces thermophilus M77

• Published in: Biochimica et biophysica acta. General subjects Vol. 1864; no. 10; p. 129681
• Main Authors: Kadowaki, Marco Antonio Seiki, Higasi, Paula Miwa Rabelo, de Godoy, Mariana Ortiz, de Araújo, Evandro Ares, Godoy, Andre Schutzer, Prade, Rolf Alexander, Polikarpov, Igor
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2020
• Subjects: AA3; AAOx; acids; active sites; Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - metabolism; alcohols; aldehydes; Aryl-alcohol oxidase; Ascomycota - chemistry; Ascomycota - enzymology; Ascomycota - metabolism; Binding Sites; calcium; Calcium - metabolism; catalytic activity; crystal structure; Crystallography, X-Ray; enzyme activity; Enzyme Stability; enzyme substrates; flavin-adenine dinucleotide; fuel production; glycosylation; hydrogen peroxide; Hydrogen Peroxide - metabolism; lignin; Models, Molecular; Myceliophthora thermophila; oxidation; Protein Conformation; small-angle X-ray scattering; structure-activity relationships; Substrate Specificity; Temperature; thermal stability; thermophilic fungi; Thermostability; Thermothelomyces thermophilus; X-ray diffraction; X-ray structure; Thermostability; Thermothelomyces thermophilus; AA3; X-ray structure; Aryl-alcohol oxidase; AAOx
• DOI: 10.1016/j.bbagen.2020.129681
• ISSN: 0304-4165; 1872-8006

Background Fungal aryl-alcohol oxidases (AAOx) are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids, with concomitant production of hydrogen peroxide (H2O2) as by-product. The H2O2 can be provided to lignin degradation pathway, a biotechnological property explored in biofuel production. In the thermophilic fungus Thermothelomyces thermophilus (formerly Myceliophthora thermophila), just one AAOx was identified in the exo-proteome. Methods The glycosylated and non-refolded crystal structure of an AAOx from T. thermophilus at 2.6 Å resolution was elucidated by X-ray crystallography combined with small-angle X-ray scattering (SAXS) studies. Moreover, biochemical analyses were carried out to shed light on enzyme substrate specificity and thermostability. Results This flavoenzyme harbors a flavin adenine dinucleotide as a cofactor and is able to oxidize aromatic substrates and 5-HMF. Our results also show that the enzyme has similar oxidation rates for bulky or simple aromatic substrates such as cinnamyl and veratryl alcohols. Moreover, the crystal structure of MtAAOx reveals an open active site, which might explain observed specificity of the enzyme. Conclusions MtAAOx shows previously undescribed structural differences such as a fully accessible catalytic tunnel, heavy glycosylation and Ca2+ binding site providing evidences for thermostability and activity of the enzymes from AA3_2 subfamily. General significance Structural and biochemical analyses of MtAAOx could be important for comprehension of aryl-alcohol oxidases structure-function relationships and provide additional molecular tools to be used in future biotechnological applications. •MtAAOx has higher oxidative activity at neutral conditions.•MtAAOx crystal structure was determined at 2.6 Å resolution.•A wider entrance explains similar enzymatic activity for small and bulky substrates.•MtAAOx binds Ca2+ which impacts its thermostability and activity.