Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly.

The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of ß-d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-ß-d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17 °C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.

Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes Aspergillus nidulans and Aspergillus oryzae.

A gene encoding a pyranose 2-oxidase (POx; pyranose/oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) was identified in the genome of the ascomycete Aspergillus nidulans. Attempts to isolate POx directly from A. nidulans cultures or to homologously overexpress the native POx (under control of the constitutive gpdA promoter) in A. nidulans were unsuccessful. cDNA encoding POx was synthesized from mRNA and expressed in Escherichia coli, and the enzyme was subsequently purified and characterized. A putative pyranose 2-oxidase-encoding gene was also identified in the genome of Aspergillus oryzae. The coding sequence was synthetically produced and was also expressed in E. coli. Both purified enzymes were shown to be flavoproteins consisting of subunits of 65 kDa. The A. nidulans enzyme was biochemically similar to POx reported in literature. From all substrates, the highest catalytic efficiency was found with D-glucose. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones and 2,6-dichloroindophenol. As judged by the catalytic efficiencies (k (cat)/k(m)), some of these quinone electron acceptors are better substrates for pyranose oxidase than oxygen. The enzyme from A. oryzae was physically similar but showed lower kinetic constants compared to the enzyme from A. nidulans. Distinct differences in the stability of the two enzymes may be attributed to a deletion and an insertion in the sequence, respectively.

Overexpression of a bacterial chymotrypsin: application for L-amino acid ester hydrolysis.

In this work, a reliable protocol was designed to rapidly express and purify a microbial chymotrypsin(ogen) as a useful alternative to using animal proteases. The cDNA encoding for chymotrypsinogen from the deuteromycete Metarhizium anisopliae (chy1) was overexpressed in an Origami2(DE3) E. coli strain deficient in thioredoxin reductase and glutathione reductase activities, thus possibly allowing disulfide exchange. By using a quick purification protocol, in which the hexahistidine tag was added at the C-terminal end of the protease, the recombinant CHY1 protein could be purified in a single step on an Ni-NTA column as a mixture of 19.5- and 15-kDa mature active forms and did not require further activation/maturation steps. This expression and purification procedure offers an easier and faster means of producing recombinant CHY1 chymotrypsin than that previously described for Pichia pastoris. The kinetic properties could be characterized and CHY1 chymotrypsin was demonstrated to efficiently catalyze N-acetylated L-phenylalanine and L-tyrosine methyl ester hydrolysis.

Importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase.

Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldopyranoses present in lignocellulose to 2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment ((454)FSY(456)) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp(452), Phe(454) and Tyr(456)) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.

Heterologous expression of an Agaricus meleagris pyranose dehydrogenase-encoding gene in Aspergillus spp. and characterization of the recombinant enzyme.

Pyranose dehydrogenase (PDH) is a flavin-dependant sugar oxidoreductase found in the family Agaricaceae, basidiomycetes that degrade lignocellulose-rich forest litter, and is catalytically related to the fungal enzymes pyranose 2-oxidase and cellobiose dehydrogenase. It has broad substrate specificity and displays similar activities with most sugar constituents of lignocellulose including disaccharides and oligosaccharides, a number of (substituted) quinones, and metal ions are suitable electron acceptors rather than molecular oxygen. In contrast to pyranose 2-oxidase and cellobiose dehydrogenase, which oxidize regioselectively at C-2 and C-1, respectively, PDH is capable of oxidation on C-1 to C-4 as well as double oxidations, depending on the nature of the substrate. This makes it a very interesting enzyme for biocatalytic applications, as many of the reaction products are otherwise unaccessible by chemical or enzymatic means. PDH was characterized in detail in a limited number of fungi, and the first encoding genes were isolated only recently. We report here, for the first time, the heterologous expression of one of these genes, encoding the major PDH protein in Agaricus meleagris, in the filamentous fungi Aspergillus nidulans, and Aspergillus niger.

Pyranose 2-oxidase from Phanerochaete chrysosporium--expression in E. coli and biochemical characterization.

The presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in yields of approximately 270 mg/l medium. The recombinant enzyme was provided with an N-terminal T7-tag and a C-terminal His(6)-tag to facilitate simple one-step purification. We obtained an apparently homogenous enzyme preparation with a specific activity of 16.5 U/mg. Recombinant P2Ox from P. chrysosporium was characterized in some detail with respect to its physical and catalytic properties, both for electron donor (sugar substrates) and - for the first time - alternative electron acceptors (1,4-benzoquinone, substituted quinones, 2,6-dichloroindophenol and ferricenium ion). As judged from the catalytic efficiencies k(cat)/K(m), some of these alternative electron acceptors are better substrates than oxygen, which might have implications for the proposed in vivo function of pyranose 2-oxidase.

A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications.

In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 172], the variant oxidizes both sugars equally well [(k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower K(M) values and approximately ten-fold higher k(cat) values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60 degrees C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D-glucose/D-galactose mixtures at both 30 and 50 degrees C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 A resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type.

Engineering of pyranose 2-oxidase: improvement for biofuel cell and food applications through semi-rational protein design.

Pyranose 2-oxidase (P2Ox) has several proposed biotechnological applications such as a bio-component in biofuel cells or for carbohydrate transformations. To improve some of the catalytic properties of P2Ox from Trametes multicolor, we selected a semi-rational approach of enzyme engineering, saturation mutagenesis of active-site residues and subsequent screening of mutant libraries for improved activity. One of the active-site mutants with improved catalytic characteristics identified was V546C, which showed catalytic constants increased by up to 5.7-fold for both the sugar substrates (D-glucose and D-galactose) and alternative electron acceptors (1,4-benzoquinone, BQ and ferricenium ion, Fc(+)], albeit at the expense of increased Michaelis constants. By combining V546C with other amino acid replacements, we obtained P2Ox variants that are of interest for biofuel cell applications due to their increased k(cat) for both BQ and Fc(+), e.g., V546C/E542K showed 4.4- and 17-fold increased k(cat) for BQ compared to the wild-type enzyme when D-glucose and D-galactose, respectively, were the saturating substrates, while V546C/T169G showed approx. 40- and 50-fold higher k(cat) for BQ and Fc(+), respectively, with D-galactose in excess. This latter variant also shows significantly modulated sugar substrate selectivity, due to an increase in k(cat)/K(M) for D-galactose and a decrease in k(cat)/K(M) for D-glucose when oxygen is the electron acceptor, as well as improved catalytic efficiencies for d-galactose, regardless of the electron acceptor used. While the wild-type enzyme strongly prefers D-glucose over D-galactose as its substrate, V546C/T169G converts both sugars equally well as was shown by the kinetic constants determined as well as by biotransformation experiments.