No abstract

Natural carbohydrate polymers such as starch, cellulose, and chitin provide renewable alternatives to fossil fuels as a source for fuels and materials. As such, there is considerable interest in their conversion for industrial purposes, which is evidenced by the established and emerging markets for products derived from these natural polymers. In many cases, this is achieved via industrial processes that use enzymes to break down carbohydrates to monomer sugars. One of the major challenges facing large-scale industrial applications utilizing natural carbohydrate polymers is rooted in the fact that naturally occurring forms of starch, cellulose, and chitin can have tightly packed organizations of polymer chains with low hydration levels, giving rise to crystalline structures that are highly recalcitrant to enzymatic degradation. The topic of this review is oxidative cleavage of carbohydrate polymers by lytic polysaccharide monooxygenases (LPMOs). LPMOs are copper-dependent enzymes (EC 1.14.99.53–56) that, with glycoside hydrolases, participate in the degradation of recalcitrant carbohydrate polymers. Their activity and structural underpinnings provide insights into biological mechanisms of polysaccharide degradation.

Background: ␤-Glucosidases hydrolyze the ␤-linkage between two adjacent molecules in oligomers of glucose. Results: We report the structure and biochemical characterization of Cel3A from Hypocrea jecorina. Conclusion:We determine the structures of Cel3A from protein expressed in two different expression hosts and compare them. Significance: The structures give new insights into protein glycosylations, stability, and ligand binding in GH3 ␤-glucosidases.

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese. Little is known about metal selection by LOX and the adjustment of the redox potentials of their protein-bound catalytic metals. Thirteen three-dimensional structures of animal, plant, and prokaryotic FeLOX are available, but none of MnLOX. The MnLOX of the most important plant pathogen, the rice blast fungus Magnaporthe oryzae (Mo), was expressed in Pichia pastoris. Mo-MnLOX was deglycosylated, purified to homogeneity, and subjected to crystal screening and x-ray diffraction. The structure was solved by sulfur and manganese single wavelength anomalous dispersion to a resolution of 2.0 Å. The manganese coordinating sphere is similar to iron ligands of coral 8R-LOX and soybean LOX-1 but is not overlapping. The Asn-473 is positioned on a short loop (Asn-Gln-Gly-Glu-Pro) instead of an ␣-helix and forms hydrogen bonds with Gln-281. Comparison with FeLOX suggests that Phe-332 and Phe-525 might contribute to the unique suprafacial hydrogen abstraction and oxygenation mechanism of Mo-MnLOX by controlling oxygen access to the pentadiene radical. Modeling suggests that Arg-525 is positioned close to Arg-182 of 8R-LOX, and both residues likely tether the carboxylate group of the substrate. An oxygen channel could not be identified. We conclude that MoMnLOX illustrates a partly unique variation of the structural theme of FeLOX. Lipoxygenases (LOX)3 are iron-or manganese-containing dioxygenases that oxidize polyunsaturated fatty acids containing one or more 1Z,4Z-pentadiene units to hydroperoxides (1, 2). These hydroperoxides are precursors of signal molecules in animals, plants, and fungi. They may take part in inflammation, asthma, cancer development, and the chemical warfare between plants, fungi, and other microorganisms (3, 4). The LOX mechanism is initiated with hydrogen abstraction from a bis-allylic carbon of the 1Z,4Z-pentadiene of fatty acids. This is followed by oxygen insertion, which usually produces cis-transconjugated hydroperoxy fatty acids (1, 2). Plant, mammals, and a few prokaryotes express FeLOX, whereas both MnLOX and FeLOX occur in plant pathogenic fungi (5-8).All LOX belong to the same gene family, but plant FeLOX, mammalian FeLOX, and fungal FeLOX and MnLOX form separate subfamilies (5, 8). The prototype MnLOX is secreted by the take-all fungus of wheat, Gaeumannomyces graminis (7). The evolution of this enzyme, 13R-MnLOX, and five members of the MnLOX subfamily are illustrated in a phylogenetic tree together with pro-and eukaryotic LOX, including fungal FeLOX (Fig. 1A).The three-dimensional structures of 11 eukaryotic and two prokaryotic FeLOX are available. These are four structures of soybean LOX (sLOX-1, LOX-3, VLX-B, and VLX-D) (9 -13), three human LOX (15-LOX-2, 12S-LOX, and 5-LOX) (2, ...

For decades, the enzymes of the fungus have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all cellulose-degrading enzymes are available, except for LPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that nativeLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of LPMO9A containing 21 residues of the predicted linker was expressed at levels sufficient for analysis. Here, using structural, spectroscopic, and biochemical analyses, we found that this truncated variant exhibited reduced binding to and activity on cellulose compared with the full-length enzyme. Importantly, a 0.95-Å resolution X-ray structure of truncatedLPMO9A revealed that the linker forms an integral part of the catalytic domain structure, covering a hydrophobic patch on the catalytic AA9 module. We noted that the oxidized catalytic center contains a Cu(II) coordinated by two His ligands, one of which has a His-brace in which the His-1 terminal amine group also coordinates to a copper. The final equatorial position of the Cu(II) is occupied by a water-derived ligand. The spectroscopic characteristics of the truncated variant were not measurably different from those of full-length LPMO9A, indicating that the presence of the CBM1 module increases the affinity ofLPMO9A for cellulose binding, but does not affect the active site.