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

Author(s)

Seiki Kadowaki, Marco Antonio ^[1] ; Rabelo Higasi, Paula Miwa ^[1] ; de Godoy, Mariana Ortiz ^[1] ; de Araujo, Evandro Ares ^[1] ; Godoy, Andre Schutzer ^[1] ; Prade, Rolf Alexander ^{[2, 3]} ; Polikarpov, Igor ^[1]

Affiliation

^[1] Univ Sao Paulo, Sao Carlos Inst Phys, Av Trabalhador Sao Carlense 400, BR-13566590 Sao Carlos, SP - Brazil

^[2] Oklahoma State Univ, Dept Microbiol & Mol Genet, Stillwater, OK 74078 - USA

^[3] Oklahoma State Univ, Dept Biochem & Mol Biol, Stillwater, OK 74078 - USA

• Document type: Journal article
• Source: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS; v. 1864, n. 10 OCT 2020.
• Web of Science Citations: 0

Abstract

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 angstrom 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. (AU)

• FAPESP's process: 15/13684-0 - Structural and functional studies of enzymes that participate in complex carbohydrates synthesis and degradation
• Grantee: Igor Polikarpov
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 11/20505-4 - Two important classes of glycosyl hydrolases: functional studies and structural analysis
• Grantee: Marco Antonio Seiki Kadowaki
• Support Opportunities: Scholarships in Brazil - Post-Doctoral