Bioinformatic Analysis of Lytic Polysaccharide Monooxygenases Reveals the Pan-Families Occurrence of Intrinsically Disordered C-Terminal Extensions

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes secreted by many organisms and viruses. LPMOs catalyze the oxidative cleavage of different types of polysaccharides and are today divided into eight families (AA9–11, AA13–17) within the Auxiliary Activity enzyme class of the CAZy database. LPMOs minimal architecture encompasses a catalytic domain, to which can be appended a carbohydrate-binding module. Intriguingly, we observed that some LPMO sequences also display a C-terminal extension of varying length not associated with any known function or fold. Here, we analyzed 27,060 sequences from different LPMO families and show that 60% have a C-terminal extension predicted to be intrinsically disordered. Our analysis shows that these disordered C-terminal regions (dCTRs) are widespread in all LPMO families (except AA13) and differ in terms of sequence length and amino-acid composition. Noteworthily, these dCTRs have so far only been observed in LPMOs. LPMO-dCTRs share a common polyampholytic nature and an enrichment in serine and threonine residues, suggesting that they undergo post-translational modifications. Interestingly, dCTRs from AA11 and AA15 are enriched in redox-sensitive, conditionally disordered regions. The widespread occurrence of dCTRs in LPMOs from evolutionarily very divergent organisms, hints at a possible functional role and opens new prospects in the field of LPMOs.

The crystal structure of CbpD clarifies substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases

“…A number of these post-transitionally modified residues are also found in the AA10-GbpA2 domain interface of CbpD, likely encouraging a more elongated overall conformation, similar to the observed solution scattering conformation. Additionally, a recent bioinformatics analysis of 27 000 LPMOs found that most of the eight LPMO AA families are enriched in Ser and Thr residues in C-terminal regions that are prime candidates for PTM, specifically O-glycosylation (Tamburrini et al, 2021), which may affect secretion (Vorkapic et al, 2019). Of note, the Tma12 structure has an N-glycosylation at Asn158, structurally equivalent to Ser136 in CbpD, which has been identified as phosphorylated when CbpD is expressed and secreted by P. aeruginosa (Ouidir et al, 2014).…”

Purification and Structural Characterization of the Auxiliary Activity 9 Native Lytic Polysaccharide Monooxygenase from Thermoascus aurantiacus and Identification of Its C1- and C4-Oxidized Reaction Products

“…Contrary to cellulases which, as glycoside hydrolases (GHs), catalyze the breakage of 1,4-β-glycosidic bonds in cellulose via a hydrolytic mechanism, LPMOs follow an oxidative mechanism [2][3][4]. Extensive studies in LPMOs have led to their further classification as Auxiliary Activity (AA) enzymes in the Carbohydrate Active enZymes (CAZy; www.cazy.org; accessed on 20 November 2021) database [5], where they form eight separate families (AA9-AA11, AA13-AA17) [6,7]. LPMOs of fungal origin are found in the families AA9 (formerly GH61) and AA11.…”

CbpD crystal structure adds intrigue to substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases

“…A number of these post-transitionally modified residues are also in the AA10:GbpA2 domain interface of CbpD, likely encouraging a more elongated overall conformation, similar to the observed solution scattering conformation. Additionally, a recent bioinformatics analysis of 27,000 LPMOs found that most of the eight LPMO AA families are enriched for Ser and Thr residues in C-terminal regions that are prime candidates for PTM, specifically O - glycosylation (Tamburrini et al ., 2021), which may affect secretion (Vorkapic et al ., 2019). Of note, the Tma12 structure has an N-glycosylation at Asn158, structurally equivalent to Ser136 in CbpD, which has been identified as phosphorylated when CbpD is expressed and secreted by P. aeruginosa (Ouidir et al ., 2014).…”