Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Abstract

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.

Keywords: carbohydrate degrading enzymes; gribble.

Fig. 1.

Abundance and functional environment of the cellobiohydrolase LqCel7B in the L. quadripunctata digestive system. (A) Reverse transcription, quantitative PCR (RT-qPCR) using RNA extracted from rest of the body (RB), hindgut (G), and hepatopancreas (HP). Expression levels presented are normalized to ubiquitin (Upper). Western blot of protein extracts prepared from these organs probed with purified anti-LqCel7B (Lower). (B) Scanning electron micrograph showing an obliquely sectioned hindgut packed with wood particles. (Scale bar, 50 µm.)

Fig. 2.

Structural and computational analysis of LqCel7B. (A) Overall fold of the apo LqCel7B structure with the catalytic residues highlighted in blue and a selection of the planar Trp residues that line the ligand binding tunnel in yellow. Tyr121 is shown in pink. The cellononaose ligand is docked into the LqCel7B structure from the HjCel7A structure and is shown in green (38). (B) Structural comparison of the apo LqCel7B structure and HjCel7A, with the ligand from 8CEL (38). (C) View of the −7 to −5 binding sites in two crystallographic and two simulation conformations, including the ligand from 8CEL (38) in green. Tyr121 binds directly to the ligand in the −7 glucose binding site. The color coding for Tyr121 and Trp57 is as follows: pink, apo structure conformations; gray, cellotriose LqCel7B structure; blue, MD simulation snapshot of LqCel7B with Tyr121 stacking on the −7 glucose unit; and yellow, MD simulation snapshot of LqCel7B with Tyr121 binding to the −6 glucose unit. Fig. S5E shows the cluster view of Tyr121 over the 250-ns MD simulation. (D) Cluster representations of LqCel7B and HjCel7A over a 250-ns MD simulation. HjCel7A data are taken from ref. , and the enzymes are colored by root mean square fluctuations (RMSFs), wherein red represents fluctuations up to 4 Å and blue represents the minimum fluctuations. The Root square mean deviation (RSMD) and RMSF of both enzymes are shown in Fig. S5 A–C. (E) RMSF of LqCel7B and HjCel7A (44) ligands. Error bars represent one SD from the average value measured by block averaging. (F) Time series of the exo-loop opening and closing events in LqCel7B and HjCel7A MD simulations over 250 ns with a cellononaose ligand present (33, 38). The exo-loop motions are the minimum distance between the exo loop and the corresponding loops on the other side of the tunnel: for LqCel7B, the minimum distance from the exo loop (residues 265–274) to residues 394–399 and for HjCel7A, the minimum distance from the exo loop (residues 244–253) to residues 369–373. A histogram of this time series is shown in Fig. S5D.

Fig. 3.

LqCel7B salt tolerance. Electrostatic potential distribution on the solvent accessible surface of Limnoria quadripunctata LqCel7B (A), Chelura terebrans GH7 (B), Daphnia magna GH7 (C), and Hypocrea jecorina Cel7A (D). Electrostatic potential between −7 kT/e and 7 kT/e was shown as a colored gradient from red (acidic) to blue (basic). (E) pI clusters showing GH7s from three GH7 L. quadripunctata enzymes grouped together with the C. terebrans ortholog (red square) as a unique cluster at low pI value (3.8 < pI < 3.9). (F) Hydrolytic activity of LqCel7B as a function of sodium chloride concentration expressed as reducing sugar released from PASC and Avicel. Asterisks represent statistically significant values following one-way ANOVA DSF (*P < 0.01).