Bioinformatic characterization of type-specific sequence and structural features in auxiliary activity family 9 proteins

Abstract

BackgroundDue to the impending depletion of fossil fuels, it has become important to identify alternative energy sources. The biofuel industry has proven to be a promising alternative. However, owing to the complex nature of plant biomass, hence the degradation, biofuel production remains a challenge. The copper-dependent Auxiliary Activity family 9 (AA9) proteins have been found to act synergistically with other cellulose-degrading enzymes resulting in an increased rate of cellulose breakdown. AA9 proteins are lytic polysaccharide monooxygenase (LPMO) enzymes, otherwise known as polysaccharide monooxygenases (PMOs). They are further classified as Type 1, 2 or 3 PMOs, depending on the different cleavage products formed. As AA9 proteins are known to exhibit low sequence conservation, the analysis of unique features of AA9 domains of these enzymes should provide insights for the better understanding of how different AA9 PMO types function.ResultsBioinformatics approaches were used to identify features specific to the catalytic AA9 domains of each type of AA9 PMO. Sequence analysis showed the N terminus to be highly variable with type-specific inserts evident in this region. Phylogenetic analysis was performed to cluster AA9 domains based on their types. Motif analysis enabled the identification of sub-groups within each AA9 PMO type with the majority of these motifs occurring within the highly variable N terminus of AA9 domains. AA9 domain structures were manually docked to crystalline cellulose and used to analyze both the type-specific inserts and motifs at a structural level. The results indicated that these regions influence the AA9 domain active site topology and may contribute to the regioselectivity displayed by different AA9 PMO types. Physicochemical property analysis was performed and detected significant differences in aromaticity, isoelectric point and instability index between certain AA9 PMO types.ConclusionsIn this study, a type-specific characterisation of AA9 domains was performed using various bioinformatics approaches. These highly variable proteins were found to have a greater degree of conservation within their respective types. Type-specific features were identified for AA9 domains, which could be observed at a sequence, structural and physicochemical level. This provides a basis under which to identify and group new AA9 LPMOs in future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0655-2) contains supplementary material, which is available to authorized users.

Structural Features on the Substrate-Binding Surface of Fungal Lytic Polysaccharide Monooxygenases Determine Their Oxidative Regioselectivity

“…Just like N ‐glycans, aromatic surface residues are believed to be determinants of LPMO regioselectivity . Indeed, these might help in accurately directing the oxidative force of the enzyme by influencing the position of the substrate in a very subtle, yet crucial manner .…”

“…Structural features on these loops, like N ‐glycans and aromatic residues are suggested to play a role in directing the oxidative power of LPMOs . The importance for C4‐oxidation of a subdomain of about 10–12 residues on the L2 loop has been demonstrated .…”

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars

“…110 This classification was further refined by Moses et al in a bioinformatic study and was included as three separate clusters (6, 7, and 8) in the larger structural classification of all LPMOs defined by Vaaje-Kolstad and co-workers in a review on the structural diversity of LPMOs. 10910 Although there are large structural variations among AA9 LPMOs, these differences are localized to two regions, the L2 and L3 regions (colored yellow and red, respectively, in Figure 12). In type 1 AA9s, both the L2 and the L3 regions are shorter than those observed in type 2 and type 3 LPMOs.…”

High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain

“…6), has the same approximate location as the N-linked glycosylations of the other three Type-3 AA9 LPMOs (HjLPMO9B (19), TaLPMO9A (9), and NcLPMO9M (32)). Interestingly, these glycans have the same spatial location as the insert region II in Type-2 LPMOs, which is the signature sequence of these strict C4 oxidizing LPMOs (11,32,38). Notably, glycosylations are usually treated enzymatically prior to crystallization, thus what can be visualized in structure models are most likely remnants of much larger glycans.…”