Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases

Abstract

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases.

Figure 1

(A) Graphical representation of the conformations of a 5-membered ring according to the Cremer–Pople angle ϕ. (B) Conformational FEL of isolated α-l-arabinofuranose. Conformations observed in Michaelis complexes of α-l-arabinofuranosidases are represented with a red star (PDB 2VRQ and 1QW9 for GH51 and PDB 6SXR, this work, for GH54). The conformational region having an equatorial O2 is shaded. (C) Conformational FEL of α-l-arabinofuranose-configured cyclophellitol (1), aziridine (2), and cyclic sulfate (6).

Figure 2

(A) Koshland double-displacement mechanism employed by retaining α-l-arabinofuranosidases, as proposed for GH51 and GH54, showing the conformational reaction itinerary including the (left-to-right) Michaelis complex, transition state 1, covalent substrate-enzyme intermediate, transition state 2, and the hydrolyzed product. (B) Chemical structures of putative α-l-arabinofuranosidase inhibitors 1, 2, 3, and 6 and ABPs 4 and 5.

Scheme 1. Synthesis of l-Arabinofuranose-Configured Cyclopentene 15

Reagents and conditions: (a) (1S)-(+)-10-camphorsulfonic acid, CH₃CN, 50 °C, 300 mbar, 2.5 h; (b) BnBr, NaH, TBAI, DMF, 0 °C, rt, 18 h, 74% over two steps; (c) BH₃·THF, Bu₂BOTf, DMF, 0 °C, 15 min, 90%; (d) I₂, TPP, THF, reflux, 3 h, 79%; (e) activated Zn powder, THF, 35 °C, 2 h, 84%; (f) Ph₃PCH₃Br, n-BuLi, THF, −78 to −20 °C for 1 h, then rt, 18 h, 73%; (g) Grubb’s II cat., DCM, reflux, 18 h, 90%; (h) DDQ, DCM, 0 °C, rt, 2 h, 86%; (i) Bu₃SnMeI, KH, dibenzo-18-crown-6, THF, 0 °C, rt, 18 h, 91%; (j) n-BuLi, THF, −78 °C to rt, 18 h, 68%.

Scheme 2. Synthesis of Epoxides

Reagents and conditions: (a) m-CPBA, DCM, 50 °C, 18 h, 62%, 3.4:1 of 16:17; (b) m-CPBA, DCM, 0 °C, 4 days, 91%, 4.3:1 of 16/17; (c) m-CPBA, DCM, 50 °C, 18 h, 62%, 1:2 of 19/20; (d) H₂, Pd(OH)₂, MeOH, 18 h, 50%.

Scheme 3. Synthesis of α-l-Aziridines 2–5

Reagents and conditions: (a) BnBr, NaH, TBAI, DMF, rt, 18 h, 78%; (b) NaN₃, LiClO₄, DMF, 100 °C, 18 h, 77%; (c) TsCl, DMAP, TEA, DCM, 0 °C, 18 h, 50%; (d) TPP, DIPEA, THF/H₂O, reflux, 1.5 h, 56%; (e) Li, NH₃, −60 °C, 1 h, 66%; (f) 8-azidooctyl triflate, DIPEA, DCM, 0 °C to rt, 18 h, 57%; (g) Na, NH₃, t-BuOH, −60 °C, 1 h, 95%; (h) Cy5-Osu or biotin-OSu, DIPEA, DMF, 18 h, 4: 56% and 5: 19%.

Scheme 4. Synthesis of Cyclic Sulfate 6

Reagents and conditions: (a) NaIO₄, RuCl₃·3H₂O, EtOAc/CH₃CN/H₂O, 0°C, 3 h, 48%; (b) (i) SOCl₂, Et₃N, DCM, 0 °C, 30 min, (ii) NaIO₄, RuCl₃·3H₂O, EtOAc/CH₃CN/H₂O, 0 °C, 3 h, 51%; (c) H₂, Pd(OH)₂, MeOH, 18 h, 24%.

Figure 3

Crystal structures of complexes between inhibitors 2 (B, D, green) and 6 (A, C, purple), and GsGH51 (A, B, blue) and AkAbfB (C, D, yellow). 2F₀ – F_c electron density is shown for both the ligand and the catalytic nucleophile as a gray mesh contoured at 2σ. The polypeptide is shown in cartoon form with active site residues shown as sticks. Apparent hydrogen bonding interactions are shown as dotted yellow lines.

Figure 4

Activity-based protein profiling of fungal secretomes with ABPs 4 and 5. (A) Fluorescence imaging of the secretome isolated from A. niger grown on arabinan, stained with ABP 4, and treated with (PNG+) or without (PNG-) PNGaseF under denaturing conditions prior to separation on an 8.75% SDS-PAGE gel. L indicates the ladder lanes. (B) Label-free quantification of the top eight proteins pulled down from the A. niger arabinan secretome. For each protein (identified by NRRL3 number and common name), integrated peptide intensity is plotted for nonconflicting peptides from the pull-down with ABP 5 (PD, black), from the total secretome (TS, blue), and from the pull-down with ABP 5 following pretreatment with inhibitor 2 (PT, red). Error bars represent the standard deviations of three measurements. (C) Cy5 fluorescence (red) and Coomassie staining (green) of basidiomycete secretomes following staining with ABP 4 and acetone precipitation. L indicates the ladder lane. The BRFM number for the strain from which the secretome was isolated is given above each lane. (D) Plot of total spectral counts in the pull-down sample vs the ratio of spectral counts in the pull-down sample to spectral counts in the total secretome for all of the proteins for which at least 2 peptides were observed with an FDR of 1% in the pull-downs from L. menziesii (BRFM 1557), F. fomentarius (BRFM 1323), T. gibbosa (BRFM 952), and A. biennis (BRFM 1215). Points corresponding to GH51 enzymes are shown in orange, points corresponding to other putative retaining GH enzyme with peptide molecular weights >90 kDa are shown in red. The labels shown include the species abbreviation and the Mycocosm amino acid sequence number.