Substrate specificity mapping of fungal CAZy AA3_2 oxidoreductases

Background
Oxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary
activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal
AA3 glucose–methanol–choline (GMC) oxidoreductases with varying auxiliary activities are attractive
sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided
into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However,
limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which
limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities
may be discovered from the uncharacterized parts of this large subfamily.
Results
In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum
likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different
clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were
successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases
(TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl
alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose)
dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability
to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete
with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases
(ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants
governing their diverse enzymatic functions were discussed based on the AlphaFold structures.
Conclusions
The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for
future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function
and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2
oxidoreductases bring exciting prospects for biotechnological applications and broadens our
understanding of their biological functions