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Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s−1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae). [ABSTRACT FROM AUTHOR]

Xylan 1,4-β-D-xylosidase catalyzes hydrolysis of non-reducing end xylose residues from xylooligosaccharides. The enzyme is currently used in combination with β-xylanases in several large-scale processes for improving baking properties of bread dough, improving digestibility of animal feed, production of d-xylose for xylitol manufacture, and deinking of recycled paper. On a grander scale, the enzyme could find employment alongside cellulases and other hemicellulases in hydrolyzing lignocellulosic biomass so that reaction product monosaccharides can be fermented to biofuels such as ethanol and butanol. Catalytically efficient enzyme, performing under saccharification reactor conditions, is critical to the feasibility of enzymatic saccharification processes. This is particularly important for β-xylosidase which would catalyze breakage of more glycosidic bonds of hemicellulose than any other hemicellulase. In this paper, we review applications and properties of the enzyme with emphasis on the catalytically efficient β- d-xylosidase from Selenomonas ruminantium and its potential use in saccharification of lignocellulosic biomass for producing biofuels. [ABSTRACT FROM AUTHOR]

β- d-Xylosidase catalyzes hydrolysis of xylooligosaccharides to d-xylose residues. The enzyme, SXA from Selenomonas ruminantium, is the most active catalyst known for the reaction; however, its activity is inhibited by d-xylose and d-glucose ( Ki values of ∼10−2 M). Higher Ki’s could enhance enzyme performance in lignocellulose saccharification processes for bioethanol production. We report here the development of a two-tier high-throughput screen where the 1° screen selects for activity (active/inactive screen) and the 2° screen selects for a higher Ki( d-xylose) and its subsequent use in screening ∼5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, Ki( d-xylose) is threefold and Ki( d-glucose) is twofold that of wild-type SXA. C3 contains four amino acid mutations, and one of these, W145G, is responsible for most of the lost affinity for the monosaccharides. Experiments that probe the active site with ligands that bind only to subsite −1 or subsite +1 indicate that the changed affinity stems from changed affinity for d-xylose in subsite +1 and not in subsite −1 of the two-subsite active site. Trp145 is 6 Å from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite −1. [ABSTRACT FROM AUTHOR]

The catalytic domain encoded by an adenine–thymine (AT)-rich xylanase gene ( xynA) of the anaerobic fungus Orpinomyces was expressed in Hypocrea jecorina under the control of the cel7A promoter and terminator. No XynA protein was detected in H. jecorina culture supernatants when the original sequence was fused to the H. jecorina cel5A region coding for its signal peptide, carbohydrate-binding module, and hinge. Replacing the xynA (56% AT content) with a synthetic sequence containing lower AT content (39%) supported the extracellular production (150 mg l−1) of the fusion xylanase by H. jecorina. Northern analysis revealed that successful production after the decrease in AT content was related to higher levels of the xylanase-specific mRNA. Another construct with an RDKR-coding sequence inserted between the cel5A linker and the xynA catalytic domain allowed production of the fully processed active xylanase catalytic domain. Both the fusion (40 kDa) and the fully processed (28 kDa) forms displayed enzymatic properties of family 11 xylanases. Both the R and the Kex2-like KR sites were recognized during secretion, resulting in a mixture of two amino termini for the 28-kDa xylanase. The work demonstrated for the first time that glycoside hydrolases derived from anaerobic fungi can be produced by H. jecorina. [ABSTRACT FROM AUTHOR]

We report here the first three-dimensional structure of the D1 C-terminal processing protease (D1P), which is encoded by the ctpA gene. This enzyme removes the C-terminal extension of the D1 polypeptide of photosystem II of oxygenic photosynthesis. Proteolytic processing is necessary to allow the light driven assembly of the tetranuclear manganese cluster, which is responsible for photosynthetic water oxidation. The X-ray structure of the Scenedesmus obliquus enzyme has been determined at 1.8 Å resolution using the multiwavelength anomalous dispersion method. The enzyme is monomeric and is composed of three folding domains. The middle domain is topologically homologous to known PDZ motifs and is proposed to be the site at which the substrate C-terminus binds. The remainder of the substrate likely extends across the face of the enzyme, interacting at its scissile bond with the enzyme active site Ser 372 / Lys 397 catalytic dyad, which lies at the center of the protein at the interface of the three domains. [ABSTRACT FROM AUTHOR]

Lumazine synthase catalyzes the penultimate step in the synthesis of riboflavin in plants, fungi, and microorganisms. The enzyme displays two quaternary structures, the pentameric forms in yeast and fungi and the 60-meric icosahedral capsids in plants and bacteria. To elucidate the structural features that might be responsible for differences in assembly, we have determined the crystal structures of lumazine synthase, complexed with the inhibitor 5-nitroso-6-ribitylamino-2,4-pyrimidinedione, from spinach and the fungus Magnaporthe grisea to 3.3 and 3.1 Å resolution, respectively. The overall structure of the subunit and the mode of inhibitor binding are very similar in these enzyme species. The core of the subunit consists of a four-stranded parallel β-sheet sandwiched between two helices on one side and three helices on the other. The packing of the five subunits in the pentameric M. grisea lumazine synthase is very similar to the packing in the pentameric substructures in the icosahedral capsid of the plant enzyme. Two structural features can be correlated to the differences in assembly. In the plant enzyme, the N-terminal β-strand interacts with the β-sheet of the adjacent subunit, thus extending the sheet from four to five strands. In fungal lumazine synthase, an insertion of two residues after strand β1 results in a completely different orientation of this part of the polypeptide chain and this conformational difference prevents proper packing of the subunits at the trimer interface in the icosahedron. In the spinach enzyme, the b-hairpin connecting helices α4 and α5 participates in the packing at the trimer interface of the icosahedron. Another insertion of two residues at this position of the polypeptide chain in the fungal enzyme disrupts the hydrogen bonding in the hairpin, and the resulting change in conformation of this loop also interferes with proper intrasubunit contacts at the trimer interface. [ABSTRACT FROM AUTHOR]

The article presents information on cloning, expression, purification and crystallization of dihydroxybutanone phosphate synthase from Magnaporthe grisea. Dihydroxybutanone phosphate synthase (DS) catalyzes a commitment step in riboflavin biosynthesis where ribulose 5-phosphate is converted to dihydroxybutanone phosphate and formate. DS was cloned from the pathogenic fungus Magnaporthe grisea (using functional complementation of an Escherichia coli DS knockout mutant) and expressed in E. coli. Of the microbial DS homologs known, the M. grisea protein has the greatest similarity to the Vibrio harveyi and Saccharomyces cerevisiae proteins at the amino-acid sequence level.