Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Aspergillus Secretomes Using Activity-Based Probes

Abstract

Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and endo -β-1,4-xylanases in the secretomes of Aspergillus niger , by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme–substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.

An overview of activity-based probes for glycosidases

“…Schröder et al recently carried a similar analysis of xylosidases and -xylanases in Aspergillus niger secretomes using cyclophellitol-derived ABPs. The authors demonstrated by both fluorescent labeling and proteomic analyses that A. niger secreted distinct catabolic enzymes depending on the carbon source utilized during its growth [58].…”

“…Whilst monosaccharide ABPs that target retaining exoglycosidases have been relatively well explored, profiling of retaining endo-glycosidases will require more complex ABPs structures to mimic the length and/or branching of their natural substrates. Given past and recent successes in the glycosylation of fluoroglycoside [20,60], cyclophellitol-derived [58,61], and bromoketone [37] warheads, the general profiling of retaining endo-glycosidases appears to be within reach.…”

“…In our experience, cyclophellitol-derived ABPs are excellent first choice molecules for a wide range of in vitro, in cellulo and even in vivo applications. Cyclophellitol-derived ABPs typically adopt a ground state 4 H3 conformation that mimics the substrate transition state conformation for many glycosidases [48,58], meaning these probes often label with high potency, producing stable enzyme-cyclitol linkages suitable for various downstream analyses. Conversely, glycosidases that do not employ a 4 H3 transition state conformation can be less reactive towards cyclophellitol-derived ABPs, as has recently been observed with GH11 -xylanases (proposed to utilize a 2,5 B transition state conformation [58]).…”

Redesigning N-glycosylation sites in a GH3 β-xylosidase improves the enzymatic efficiency

“…4). The average distance between the corresponding Asp/Glu residues in other GH3 members, as measured from the crystal structures in PDB: 1IEX [16], 2X41 [17], 3ZYZ [18], 4I3G [19], 5AE6 [to be published], and 6Q7I [20]), is approximately 13 Å.

Fig. 4Distribution of distances between the α-carbons of the catalytic residues Asp288 and Glu491 along the last 350 ns of each simulation.

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“…Molecular dynamics simulations were performed on the BxlB in both its N1;5 and non-glycosylated forms using the NAMD package [56] with CHARMM36 [57] force field for proteins and carbohydrates, and TIP3P water model [58]. The BxlB structure was modeled using the I-TASSER server [59], with the PDB structure 6Q7I [20] (45.5% identity and 62.4% similarity to BxlB) as a template and further refined with the GalaxyWEB server [60]. The hydrogen atoms of the enzyme were added according to the protonation state predicted by the H++ server [61, 62] at pH 5.5.…”