In vitro and in vivo characterization of three Cellvibrio japonicus glycoside hydrolase family 5 members reveals potent xyloglucan backbone-cleaving functions

Abstract

BackgroundXyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved.ResultsCombined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here.ConclusionsThrough a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural–functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1039-6) contains supplementary material, which is available to authorized users.

Synthesis and application of a highly branched, mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor ofendo-xyloglucanases

“…The endo-xyloglucanases Cellvibrio japonicus GH5D (CjGH5D), 46 Cellvibrio japonicus GH74 (CjGH74), 47 Bacteroides ovatus GH5 (BoGH5), 38 and Prevotella bryantii GH5 (PbGH5) 48 were produced recombinantly and purified as previously described.…”

“…Gene overexpression and recombinant protein purification were then performed as described for the wild-type enzyme. 46 The concentration of the purified recombinant CjGH5D(E255A) was determined using an Epoch Micro-Volume Spectrophotometer System (BioTek®, USA) at 280 nm. The presence of the desired mutation was confirmed by intact protein mass spectrometry.…”

“…Linear initial-rate kinetics of 2-chloro-4-nitrophenolate release were measured at 405 nm (ε = 17.74 mM −1 cm −1 ) over 1 min in a quartz cuvette (l = 1 cm) maintained at 40°C, essentially as previously described. 46 Prior to measuring burst kinetics, the background hydrolysis rate of 235 µL of 5 mM XXXG(2F)-β DNP (5) in 50 mM phosphate buffer, pH 7.5, was measured at 40°C for 75 min (ε 405 = 11.17 mM −1 cm −1 , determined using a standard curve of absorbance as a function of dinitrophenolate concentration at the specified buffer concentration and pH conditions. To make the standard solution, 2,4-dinitrophenol was desiccated overnight in presence of phosphorus pentoxide, and thereafter dissolved in the specified buffer).…”

“…X-ray crystallography and structure solution CjGH5D (E255A) was crystallized with protein at 20 mg mL −1 in 20 mM sodium phosphate buffer, pH 7.0, containing 10% (v/v) glycerol, by employing similar conditions to those used to crystallize the wild-type protein. 46 A crystal grown from a protein stock solution supplemented with 5 mM inhibitor was harvested directly into liquid nitrogen using a CryoLoop™ (Hampton Research, Aliso Viejo, CA, USA). In addition, an unliganded crystal was soaked with 2 mM inhibitor for 45 min, and harvested similarly.…”

“…To examine the efficacy of XXXG(2F)-β-DNP (5) as an inhibitor, we incubated a high concentration (2.5 mM) of the compound with three configuration-retaining GH5 endo-(xylo)glucanases with differing specificity profiles: BoGH5 from the human gut bacterium Bacteroides ovatus, 38 PbGH5 from the ruminant bacterium Prevotella bryantii, 48 and CjGH5D from the soil saprophyte Cellvibrio japonicus. 46 The configuration-inverting GH74 endo-xyloglucanase from Cellvibrio japonicus, CjGH74, 47 which is not expected to be covalently inhibited because it uses a single step, direct water-attack mechanism, 60 was also examined as a negative control. As anticipated, we observed timedependent, 1 : 1 stoichiometric labelling of all three configuration-retaining GH5 enzymes by intact protein mass spectrometry, while CjGH74 remained entirely unmodified during a 12 h incubation (Fig.…”