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Genetically encoded FRET nanosensors were developed to measure cytosolic tryptophan steady-state levels and flux in real time. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 77 (GH77) belongs to the α-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-α-glucanotransferases, involved in maltose metabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltase from the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesis of the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transition state stabilizer) shows that GH77 Tt AMase and GH13 enzymes share the same catalytic machinery. Quantification of the enzyme's transglycosylation and hydrolytic activities revealed that Tt AMase is among the most efficient 4-α-glucanotransferases in the or-amylase superfamily. The active site contains at least seven substrate binding sites, subsites -2 and +3 favoring substrate binding and subsites -3 and +2 not, in contrast to several GH13 enzymes in which subsite +2 contributes to oligosaccharide binding. A model of a maltoheptaose (G7) substrate bound to the enzyme was used to probe the details of the interactions of the substrate with the protein at acceptor subsites +2 and +3 by site-directed mutagenesis. Substitution of the fully conserved Asp249 with a Ser in subsite +2 reduced the activity 23-fold (for G7 as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of α-amylases only up to 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13 amylases and GH77 amylomaltases. [ABSTRACT FROM AUTHOR]

Amylomaltases are 4-α-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer α-1,4-linked glucans to another acceptor, which can be the 4-OH group of an α-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum aerophilum IM2 was cloned and expressed in Escherichia coli, and the gene product (PyAMase) was characterized. PyAMase displays optimal activity at pH 6.7 and 95°C and is the most thermostable amylomaltase described to date. The thermostability of PyAMase was reduced in the presence of 2 mM dithiothreitol, which agreed with the identification of two possible cysteine disulfide bridges in a three-dimensional model of PyAMase. The kinetics for the disproportionation of malto-oligosaccharides, inhibition by acarbose, and binding mode of the substrates in the active site were determined. Acting on gelatinized food-grade potato starch, PyAMase produced a thermoreversible starch product with gelatin-like properties. This thermoreversible gel has potential applications in the food industry. This is the first report on an archaeal amylomaltase. [ABSTRACT FROM AUTHOR]

Examines the structure and substrate specificity of beta-glucosidase (CelB) from the hyperthermophilic archeon Pyrococcus furiosus. Difference in substrate specificity between two types of glycosidases; Characterization of the purified mutant CelB enzyme through mutagenesis; Cell site substitutions of CelB to increase phosphorylated galactose activity.

Examines the improvement in low-temperature catalysis in Pyrococcus furiosus (CelB) by directed evolution. Selection of CelB variants with higher activity on p-nitrophenyl-beta-delta-glucopyranoside at room temperature; Accommodation of CelB on amino acids; Residues critical to the determination of thermostability, low-temperature activity and substrate recognition.

Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant β-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the β-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3AinH. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)β-D-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A).Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 β-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn208 and Asn310. In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived fromeither host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms. [ABSTRACT FROM AUTHOR]

Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent glycosyl enzyme intermediate and a covalent intermediate in complex with an analogue of a co-substrate and show how the structures of both enzyme and substrate respond to the changes required by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile changes conformation dramatically during the reaction. Also, Gln-256 on the 250s loop is involved in orienting the substrate in the +1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity. [ABSTRACT FROM AUTHOR]

Bacterial fructansucrase enzymes belong to glycoside hydrolase family 68 and catalyze transglycosylation reactions with sucrose, resulting in the synthesis of fructooligosaccharides and/or a fructan polymer. Significant differences in fructansucrase enzyme product specificities can be observed, i.e. in the type of polymer (levan or inulin) synthesized, and in the ratio of polymer versus fructooligosaccharide synthesis. The Lactobacillus reuteri 121 inulosucrase enzyme produces a diverse range of fructooligosaccharide molecules and a minor amount of inulin polymer [with β(2–1) linkages]. The three-dimensional structure of levansucrase (SacB) of Bacillus subtilis revealed eight amino acid residues interacting with sucrose. Sequence alignments showed that six of these eight amino acid residues, including the catalytic triad (D272, E523 and D424, inulosucrase numbering), are completely conserved in glycoside hydrolase family 68. The other three completely conserved residues are located at the − 1 subsite (W271, W340 and R423). Our aim was to investigate the roles of these conserved amino acid residues in inulosucrase mutant proteins with regard to activity and product profile. Inulosucrase mutants W340N and R423H were virtually inactive, confirming the essential role of these residues in the inulosucrase active site. Inulosucrase mutants R423K and W271N were less strongly affected in activity, and displayed an altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer. Our data show that the − 1 subsite is not only important for substrate recognition and catalysis, but also plays an important role in determining the size of the products synthesized. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase family 3 (GH3) β‐glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β‐Glucosidases hydrolyse the β‐1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an Rcryst and Rfree of 0.18 and 0.22, respectively. NcCel3A is an extensively N‐glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β‐glucosidase from Rasamsonia emersonii. NcCel3A is a three‐domain protein with a number of extended loops that deepen the active‐site cleft of the enzyme. These structures characterize this subfamily of GH3 β‐glucosidases and account for the high cellobiose specificity of this subfamily. The glycoside hydrolase family 3 β‐glucosidase Cel3A from Neurospora crassa (NcCel3A) is highly specific for cellobiose. The crystal structure of NcCel3A has been solved to 2.25 Å resolution. The structure is a dimer and exhibits a high degree of N‐glycosylation. [ABSTRACT FROM AUTHOR]

For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose- degrading enzymes are available, except for HjLPMO9A, 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 native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of HjLPMO9A 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 truncated HjLPMO9Arevealed 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 HjLPMO9A, indicating that the presence of the CBM1 module increases the affinity of HjLPMO9A for cellulose binding, but does not affect the active site. [ABSTRACT FROM AUTHOR]

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase from H. jecorina (HjCel3A) with the β-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 β-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures. [ABSTRACT FROM AUTHOR]

The complex technology of converting lignocellulose to fuels such as ethanol has advanced rapidly over the past few years, and enzymes are a critical component of this technology. The production of effective enzyme systems at cost structures that facilitate commercial processes has been the focus of research for many years. Towards this end, the H. jecorina cellobiohydrolases, CEL7A and CEL6A, have been the subject of protein engineering at Genencor. Our first rounds of cellobiohydrolase engineering were directed towards improving the thermostability of both of these enzymes and produced variants of CEL7A and CEL6A with apparent melting temperatures above 70°C, placing their stability on par with that of H. jecorina CEL5A (EG2) and CEL3A (BGL1). We have now moved towards improving CEL6A- and CEL7A-specific performance in the context of a complete enzyme system under industrially relevant conditions. Achievement of these goals required development of new screening strategies and tools. We discuss these advances along with some results, focusing mainly on engineering of CEL6A. [ABSTRACT FROM AUTHOR]

We have previously shown that the hyperthermophilic glycosynthase from Sulfolobus solfataricus (Ssβ-glyE387G) can promote the synthesis of branched oligosaccharides from activated β-glycosides, at pH 6.5, in the presence of 2 M sodium formate as an external nucleophile. In an effort to increase the synthetic potential of hyperthermophilic glycosynthases, we report a new method to reactivate the Ssβ-glyE387G glycosynthase and two novel mutants in the nucleophile of the β-glycosidases from the hyperthermophilic Archaea Thermosphaera aggregans (Taβ-gly) and Pyrococcus furiosus (CelB). We describe here that, at pH 3.0 and low concentrations of sodium formate buffer, the three hyperthermophilic glycosynthases show k[sub cat] values similar to those of the wild-type enzymes and 17-fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5. Moreover, at acidic pH the three reactivated mutants have wide substrate specificity and improved efficiency in the synthetic reaction. The data reported suggest that the reactivation conditions modify the ionization state of the residue acting as an acid/base catalyst. This new reactivation method can be of general applicability on hyperthermophilic glycosynthases whose intrinsic stability allows their exploitation as synthetic tools at low pH. [ABSTRACT FROM AUTHOR]

Provides information on the Pyrococcus furiosus endo-beta-1,3-glucanase that belongs to the subfamily of laminarinase, which can be classified as 'all beta proteins.' Possibility to observe tertiary structure elements under extremely denaturing conditions; Preservation of secondary structure elements as indicated by the analysis of the protein spectral properties.