No abstract

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.

<div>Lytic polysaccharide monooxygenases (LPMOs) are enzymes that binds polysaccharides followed by an (oxidative) disruption of the polysaccharide surface, thereby boosting depolymerization. The binding process between LPMO and polysaccharide is key to the mechanism and recent investigations have established structure-function relationships for this binding, employing hyperfine coupling constants (HFCs) from EPR spectroscopy. Unfortunately, EPR does not provide direct structural data and therefore the experimental EPR parameters have been supported with parameters cal-</div><div>culated with density functional theory. Yet, calculated HFCs are extremely sensitive</div><div>to the employed computational setup. Using the LPMO Ls(AA9)A, we here quantify</div><div>the importance of several choices in the computational setup, ranging from the use</div><div>of specialized basis, the underlying structures, and the employed exchange–correlation</div><div>functional. We compare our results to both X-ray structures and experiment (EPR spectra) for Ls(AA9)A as well as to recent experimental/theoretical results for another</div><div>(AA10) family of LPMOs.</div>

Asymmetric hydrogenation catalysis by [(μ-H)₂Ru₃(μ₃-S)(CO)₇(μ-P–P*)] (P–P* = chiral diphosphine) indicates intact chiral clusters as active catalysts.

<div>Lytic polysaccharide monooxygenases (LPMOs) are enzymes that binds polysaccharides followed by an (oxidative) disruption of the polysaccharide surface, thereby boosting depolymerization. The binding process between LPMO and polysaccharide is key to the mechanism and recent investigations have established structure-function relationships for this binding, employing hyperfine coupling constants (HFCs) from EPR spectroscopy. Unfortunately, EPR does not provide direct structural data and therefore the experimental EPR parameters have been supported with parameters cal-</div><div>culated with density functional theory. Yet, calculated HFCs are extremely sensitive</div><div>to the employed computational setup. Using the LPMO Ls(AA9)A, we here quantify</div><div>the importance of several choices in the computational setup, ranging from the use</div><div>of specialized basis, the underlying structures, and the employed exchange–correlation</div><div>functional. We compare our results to both X-ray structures and experiment (EPR spectra) for Ls(AA9)A as well as to recent experimental/theoretical results for another</div><div>(AA10) family of LPMOs.</div>