Structural and functional investigation of a fungal member of carbohydrate esterase family 15 with potential specificity for rare xylans.

In plant cell walls, covalent bonds between polysaccharides and lignin increase recalcitrance to degradation. Ester bonds are known to exist between glucuronic acid moieties on glucuronoxylan and lignin, and these can be cleaved by glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15). GEs are found in both bacteria and fungi, and some microorganisms also encode multiple GEs, although the reason for this is still not fully clear. The fungus Lentithecium fluviatile encodes three CE15 enzymes, of which two have previously been heterologously produced, although neither was active on the tested model substrate. Here, one of these, LfCE15C, has been investigated in detail using a range of model and natural substrates and its structure has been solved using X-ray crystallography. No activity could be verified on any tested substrate, but biophysical assays indicate an ability to bind to complex carbohydrate ligands. The structure further suggests that this enzyme, which possesses an intact catalytic triad, might be able to bind and act on more extensively decorated xylan chains than has been reported for other CE15 members. It is speculated that rare glucuronoxylans decorated at the glucuronic acid moiety may be the true targets of LfCE15C and other CE15 family members with similar sequence characteristics.

Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii.

The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from CkXyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima. GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1-CBM22.2-Xyn10C construct, indicating a compact arrangement at room temperature.

Estimating the accuracy of calculated electron paramagnetic resonance hyperfine couplings for a lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are enzymes that bind polysaccharides followed by an (oxidative) disruption of the polysaccharide surface, thereby boosting depolymerization. The binding process between the LPMO catalytic domain and polysaccharide is key to the mechanism and establishing structure-function relationships for this binding is therefore crucial. The hyperfine coupling constants (HFCs) from EPR spectroscopy have proven useful for this purpose. Unfortunately, EPR does not provide direct structural data and therefore the experimental EPR parameters have to be supported with parameters calculated with density functional theory. Yet, calculated HFCs are extremely sensitive to the employed computational setup. Using the LPMO Ls(AA9)A catalytic domain, we here quantify the importance of several choices in the computational setup, ranging from the use of specialized basis, the underlying structures, and the employed exchange-correlation functional. We show that specialized basis sets are an absolute necessity, and also that care has to be taken in the optimization of the underlying structure: only by allowing large parts of the protein around the active site to structurally relax could we obtain results that uniformly reproduced experimental trends. We compare our results to previously published X-ray structures and experimental HFCs for Ls(AA9)A as well as to recent experimental/theoretical results for another (AA10) family of LPMOs.

Asymmetric hydrogenation of an α-unsaturated carboxylic acid catalyzed by intact chiral transition metal carbonyl clusters - diastereomeric control of enantioselectivity.

Twenty clusters of the general formula [(µ-H)2Ru3(µ3-S)(CO)7(µ-P-P*)] (P-P* = chiral diphosphine of the ferrocene-based Walphos or Josiphos families) have been synthesised and characterised. The clusters have been tested as catalysts for asymmetric hydrogenation of tiglic acid [trans-2-methyl-2-butenoic acid]. The observed enantioselectivities and conversion rates strongly support catalysis by intact Ru3 clusters. A catalytic mechanism involving an active Ru3 catalyst generated by CO loss from [(µ-H)2Ru3(µ3-S)(CO)7(µ-P-P*)] has been investigated by DFT calculations.