Discovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs

Abstract

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, revealed a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expanded the AA7-substrate range and unveiled a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses revealed unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly transferred electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuelled cellulose degradation by LPMOs without exogenous reductants. The expansion of the substrate range and the synergistic interplay with LPMOs highlight the functional diversity within AA7 and set the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.

In Silico Predictions of Ecological Plasticity Mediated by Protein Family Expansions in Early-Diverging Fungi

“…Homologs of AA7 were found in plants, fungi and fungi-like microorganisms. A huge abundance of AA7 genes was revealed in phytopathogenic fungi and Oomycota [38]. We also observed an increased occurrence of AA7 homologs in saprotrophic and entomopathogenic fungi.…”

“…The AA7 family harbours oligosaccharide flavo-oxidases which can transfer electrons to lytic polysaccharide monooxygenase and, consequently, fuel cellulose degradation [38]. Homologs of AA7 were found in plants, fungi and fungi-like microorganisms.…”

On the expansion of biological functions of lytic polysaccharide monooxygenases

“…However, in a preprint in 2016 (eventually published in 2017 (Bissaro et al ., 2017)), Bissaro and colleagues demonstrated the occurrence of an efficient peroxygenase reaction (Bissaro et al ., 2017), that is using H 2 O 2 rather than O 2 . At this time, multiple kinetics studies have shown that both apparent affinity and catalytic efficiency are several orders of magnitude higher for H 2 O 2 (Hangasky et al ., 2018; Kuusk et al ., 2018, 2019; Bissaro et al ., 2020a; Hedison et al ., 2020; Jones et al ., 2020; Kont et al ., 2020; Momeni et al ., 2021). This conclusion is further supported by synergistic studies with H 2 O 2 ‐producing redox partners (Bissaro et al ., 2017; Hegnar et al ., 2019; Kracher et al ., 2020; Momeni et al ., 2021).…”

“…At this time, multiple kinetics studies have shown that both apparent affinity and catalytic efficiency are several orders of magnitude higher for H 2 O 2 (Hangasky et al ., 2018; Kuusk et al ., 2018, 2019; Bissaro et al ., 2020a; Hedison et al ., 2020; Jones et al ., 2020; Kont et al ., 2020; Momeni et al ., 2021). This conclusion is further supported by synergistic studies with H 2 O 2 ‐producing redox partners (Bissaro et al ., 2017; Hegnar et al ., 2019; Kracher et al ., 2020; Momeni et al ., 2021). However, there is the possibility that the actual mechanistic path depends on the biological context and substrate/cosubstrate availability.…”

“…It should, however, be underscored that the identity of the reducing agent remains to be clarified for several of the biological contexts involving LPMOs (described later). In vitro studies have shown that reduction can occur via reaction with a variety of molecules (Kracher et al ., 2016; Frommhagen et al ., 2018), including small organic reductants (Vaaje‐Kolstad et al ., 2010), redox enzyme partners (Phillips et al ., 2011; Garajova et al ., 2016; Kracher et al ., 2016; Várnai et al ., 2018; Momeni et al ., 2021), lignin and their compounds (Westereng et al ., 2015), or even photoactivated molecules (Bissaro et al ., 2016, 2020a; Cannella et al ., 2016). A more controversial aspect pertains to the nature of the biological cosubstrate bringing the oxygen atom necessary for hydroxylation of either the C1 or C4 carbon of the scissile glycosidic bond (Vaaje‐Kolstad et al ., 2010; Phillips et al ., 2011), and inducing cleavage of the polysaccharide chain (Beeson et al ., 2012; Wang et al ., 2018).…”