A Mushroom P450-Monooxygenase Enables Regio- and Stereoselective Biocatalytic Synthesis of Epoxycyclohexenones

An epoxycyclohexenone (ECH) moiety occurs in natural products of both bacteria and ascomycete and basidiomycete fungi. While the enzymes for ECH formation in bacteria and ascomycetes have been identified and characterized, it remained obscure how this structure is biosynthesized in basidiomycetes. In this study, we i) identified a genetic locus responsible for panepoxydone biosynthesis in the basidiomycete mushroom Panus rudis and ii) biochemically characterized PanH, the cytochrome P450 enzyme catalyzing epoxide formation in this pathway. Using a PanH-producing yeast as a biocatalyst, we synthesized a small library of bioactive ECH compounds as a proof of concept. Furthermore, homology modeling, molecular dynamics simulation, and site directed mutation revealed the substrate specificity of PanH. Remarkably, PanH is unrelated to ECH-forming enzymes in bacteria and ascomycetes, suggesting that mushrooms evolved this biosynthetic capacity convergently and independently of other organisms.     

Production of Cellobiose Dehydrogenase from a Newly Isolated White Rot Fungus Termitomyces sp. OE147

Class I cellobiose dehydrogenases (CDHs) are extracellular hemoflavo enzymes produced at low levels by the Basidiomycetes (white rot fungi). In presence of suitable electron acceptors, e.g., cytochrome c, 2,6-dichlorophenol-indophenol, or metal ions, it oxidizes cellobiose to cellobionolactone. A stringent requirement for disaccharides makes CDH also useful for conversion of lactose to lactobionic acid, an important ingredient in pharma and detergent industry. In this work, class I CDH was produced using a newly identified white rot fungus Termitomyces sp. OE147. Four media were evaluated for CDH production, and maximum enzyme activity of 0.92 international unit (IU)/ml was obtained on Ludwig medium under submerged conditions. Statistical optimization of N source, which had significant effect on CDH production, using Box–Behnken design followed by optimization of inoculum size and age resulted in an increase in activity to 2.9 IU/ml and a productivity of ~25 IU/l/h. The nearly purified CDH exhibited high activity of 26.4 IU/mg protein on lactose indicating this enzyme to be useful for lactobionic acid synthesis. Some of the internal peptide sequences bore 100 % homology to the CDH produced in Myceliophthora thermophila. The fungal isolate was amenable to scale up, and an overall productivity of ~18 IU/l/h was obtained at 14-l level.     

Localized Deposition of Au Nanoparticles by Direct Electron Transfer through Cellobiose Dehydrogenase

Cellobiose dehydrogenase (CDH) is a fascinating extracellular fungal enzyme that consists of two domains, one carrying a flavin adenine dinucleotide (FAD) and the other a cytochrome‐type heme b group as cofactors. The two domains are interconnected by a linker and electrons can shuttle from the FAD to the heme group by intramolecular electron transfer. Electron transfer between CDH and an electrode can occur by direct electron transfer (DET) and by mediated electron transfer (MET). This characteristic makes CDH an interesting candidate for integration in systems such as biosensors and biofuel cells. Moreover, it makes CDH an alternative for the reduction of metal ions through DET and MET. In this work we have explored the localized deposition of gold on Pd substrates by CDH through DET and MET. For this purpose we exploited the advantage of scanning electrochemical microscopy (SECM) as a patterning tool. We first demonstrated that gold nanoparticles can be formed in homogenous solution. Then we showed that Au nanoparticles can also be locally formed and deposited on surfaces through DET at low pH and by MET at neutral pH using benzoquinone/hydroquinone as mediator.     

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.     

Properties of a recombinant β-glucosidase from polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis

A β-glucosidase (BglA, EC 3.2.1.21) gene from the polycentric anaerobic fungus Orpinomyces PC-2 was cloned and sequenced. The enzyme containing 657 amino acid residues was homologous to certain animal, plant, and bacterial β-glucosidases but lacked significant similarity to those from aerobic fungi. Neither cellulose- nor protein-binding domains were found in BglA. When expressed in Saccharomyces cerevisiae, the enzyme was secreted in two forms with masses of about 110 kDa and also found in two forms associated with the yeast cells. K _m and V _max values of the secreted BglA were 0.762 mM and 8.20 μmol/(min·mg), respectively, with p-nitrophenyl-β-d-glucopyranoside (pNPG) as the substrate and 0.310 mM and 6.45 μmol/(min·mg), respectively, for the hydrolysis of cellobiose. Glucose competitively inhibited the hydrolysis of pNPG with a K _i of 3.6 mM. β-Glucosidase significantly enhanced the conversion of cellulosic materials into glucose by Trichoderma reesei cellulase preparations, demonstrating its potential for use in biofuel and feedstock chemical production. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may be suitable.     

Lignin biodegradation by the ascomyceteChrysonilia sitophila

The lignin biodegradation process has an important role in the carbon cycle of the biosphere. The study of this natural process has developed mainly with the use of basidiomycetes in laboratory investigations. This has been a logical approach since most of the microorganisms involved in lignocellulosic degradation belong to this class of fungi. However, other microorganisms such as ascomycetes and also some bacteria, are involved in the lignin decaying process. This work focuses on lignin biodegradation by a microorganism belonging to the ascomycete class,Chrysonilia sitophila. Lignin peroxidase production and characterization, mechanisms of lignin degradation (lignin model compounds and lignin in wood matrix) and biosynthesis of veratryl alcohol are outstanding. Applications of C.sitophila for effluent treatment, wood biodegradation and single-cell protein production are also discussed.     

β-d-Xylosidase from Geobacillus thermoleovorans IT-08: Biochemical Characterization and Bioinformatics of the Enzyme

The gene encoding a thermostable β-d-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M _r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6–8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k _cat/K _M) of 0.0048?±?0.0010 s^?1 mM^?1 on p-nitrophenyl-β-d-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.     

CelF of Orpinomyces PC-2 has an intron and encodes a cellulase (CelF) containing a carbohydrate-binding module

A cDNA, designated celF, encoding a cellulase (CelF) was isolated from the anaerobic fungus Orpinomyces PC-2. The open reading frame contains regions coding for a signal peptide, a carbohydrate-binding module (CBM), a linker, and a catalytic domain. The catalytic domain was homologous to those of CelA and CelC of the same fungus and to that of the Neocallimastix patriciarum CELA, but CelF lacks a docking domain, characteristic for enzymes of cellulosomes. It was also homologous to the cellobiohydrolase IIs and endoglucanases of aerobic organisms. The gene has a 111-bp intron, located within the CBM-coding region. Some biochemical properties of the purified recombinant enzyme are described. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

A Novel Multifunctional α-Amylase from the Thermophilic Fungus Malbranchea cinnamomea: Biochemical Characterization and Three-Dimensional Structure

A novel α-amylase (McAmyA) from the thermophilic fungus, Malbranchea cinnamomea was purified, characterized and crystallized in the present study. McAmyA was purified to apparent homogeneity with a molecular mass of 60.3 kDa on SDS-PAGE. The enzyme exhibited maximal activity at pH 6.5 and was stable within pH 5.0–10.0. It was most active at 65 °C and was stable up to 50 °C. McAmyA was capable of hydrolyzing amylose, starch, amylopectin, pullulan, cyclodextrins and maltooligosaccharides. The full-length cDNA of an α-amylase gene (McAmyA) from the strain was cloned. McAmyA consisted of a 1,476-bp open reading frame encoding 492 amino acids. It displayed the highest amino acid sequence homology (less than 60 %) with the reported α-amylases. The crystal structure of McAmyA was solved at a resolution of 2.25 Å (PDB code 3VM7). The overall structure of McAmyA reveals three domains with ten α helices and 14 β strands, and the putative catalytic residues are positioned at domain A with somewhat different secondary structural circumstances compared with typical α-amylases.     

Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium

The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 μM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e⁻/H⁺ acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.     

Direct electrochemistry of Phanerochaete chrysosporium cellobiose dehydrogenase covalently attached onto gold nanoparticle modified solid gold electrodes

Achieving efficient electrochemical communication between redox enzymes and various electrode materials is one of the main challenges in bioelectrochemistry and is of great importance for developing electronic applications. Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome composed of a catalytic FAD containing dehydrogenase domain (DH(CDH)), a heme b containing cytochrome domain (CYT(CDH)), and a flexible linker region connecting the two domains. Efficient direct electron transfer (DET) of CDH from the basidiomycete Phanerochaete chrysosporium (PcCDH) covalently attached to mixed self-assembled monolayer (SAM) modified gold nanoparticle (AuNP) electrode is presented. The thiols used were as follows: 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid (4-MBA), 4-mercaptophenol (4-MP), 11-mercapto-1-undecanamine (MUNH(2)), 11-mercapto-1-undecanoic acid (MUCOOH), and 11-mercapto-1-undecanol (MUOH). A covalent linkage between PcCDH and 4-ATP or MUNH(2) in the mixed SAMs was formed using glutaraldehyde as cross-linker. The covalent immobilization and the surface coverage of PcCDH were confirmed with surface plasmon resonance (SPR). To improve current density, AuNPs were cast on the top of polycrystalline gold electrodes. For all the immobilized PcCDH modified AuNPs electrodes, cyclic voltammetry exhibited clear electrochemical responses of the CYT(CDH) with fast electron transfer (ET) rates in the absence of substrate (lactose), and the formal potential was evaluated to be +162 mV vs NHE at pH 4.50. The standard ET rate constant (k(s)) was estimated for the first time for CDH and was found to be 52.1, 59.8, 112, and 154 s(-1) for 4-ATP/4-MBA, 4-ATP/4-MP, MUNH(2)/MUCOOH, and MUNH(2)/MUOH modified electrodes, respectively. At all the mixed SAM modified AuNP electrodes, PcCDH showed DET only via the CYT(CDH). No DET communication between the DH(CDH) domain and the electrode was found. The current density for lactose oxidation was remarkably increased by introduction of the AuNPs. The 4-ATP/4-MBA modified AuNPs exhibited a current density up to 30 μA cm(-2), which is ～70 times higher than that obtained for a 4-ATP/4-MBA modified polycrystalline gold electrode. The results provide insight into fundamental electrochemical properties of CDH covalently immobilized on gold electrodes and promote further applications of CDHs for biosensors, biofuel cells, and bioelectrocatalysis.     

p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases

Phagocytes such as neutrophils play a vital role in host defense against microbial pathogens. The anti-microbial function of neutrophils is based on the production of superoxide anion (O₂^•-), which generates other microbicidal reactive oxygen species (ROS) and release of antimicrobial peptides and proteins. The enzyme responsible for O₂^•- production is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox, also called NOX2, which together form the cytochrome b₅₅₈) and four cytosolic proteins (p47phox, p67phox, p40phox and a GTPase Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate agents. This process is dependent on the phosphorylation of the cytosolic protein p47phox. p47phox is a 390 amino acids protein with several functional domains: one phox homology (PX) domain, two src homology 3 (SH3) domains, an auto-inhibitory region (AIR), a proline rich domain (PRR) and has several phosphorylated sites located between Ser303 and Ser379. In this review, we will describe the structure of p47phox, its phosphorylation and discuss how these events regulate NADPH oxidase activation.     

Xylitol Production by Genetically Engineered Trichoderma reesei Strains Using Barley Straw as Feedstock

Xylitol, a naturally occurring five-carbon sugar alcohol derived from d-xylose, is currently in high demand by industries. Trichoderma reesei, a prolific industrial cellulase and hemicellulase producing fungus, is able to selectively use d-xylose from hemicelluloses for xylitol production. The xylitol production by T. reesei can be enhanced by genetic engineering of blocking further xylitol metabolism in the d-xylose pathway. We have used two different T. reesei strains which are impaired in the further metabolism of xylitol including a single mutant in which the xylitol dehydrogenase gene was deleted (?xdh1) and a double mutant where additionally l-arabinitol-4-dehydrogenase, an enzyme which can partially compensate for xylitol dehydrogenase function, was deleted (?lad1?xdh1). Barely straw was first pretreated using NaOH and Organosolv pretreatment methods. The highest xylitol production of 6.1 and 13.22 g/L was obtained using medium supplemented with 2 % Organosolv-pretreated barley straw and 2 % d-xylose by the ?xdh1 and ?lad1?xdh1 strains, respectively.     

Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer-modified nanoporous gold electrodes

Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (±0.4), 16.0 (±0.1) and 2.0 (±0.1) μM were obtained for CtCDH-modified lactose and glucose biosensors and GOx-modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells composed of GOx- and CtCDH-modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase as cathodes, in biofuel cells. A maximum power density of 41 μW/cm² was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O₂-saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl).     

Regioselective Dichlorination of a Non‐Activated Aliphatic Carbon Atom and Phenolic Bismethylation by a Multifunctional Fungal Flavoenzyme

The regioselective functionalization of non‐activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono‐ and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O‐methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH₂‐dependent enzyme is uniquely capable of the stepwise gem‐dichlorination of a non‐activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.     

The pyrroloquinoline quinone biosynthesis pathway revisited: A structural approach

Background

Vibrio carchariae chitinase A (EC3.2.1.14) is a family-18 glycosyl hydrolase and comprises three distinct structural domains: i) the amino terminal chitin binding domain (ChBD); ii) the (α/β)₈ TIM barrel catalytic domain (CatD); and iii) the α + β insertion domain. The predicted tertiary structure of V. carchariae chitinase A has located the residues Ser33 & Trp70 at the end of ChBD and Trp231 & Tyr245 at the exterior of the catalytic cleft. These residues are surface-exposed and presumably play an important role in chitin hydrolysis.

Results

Point mutations of the target residues of V. carchariae chitinase A were generated by site-directed mutagenesis. With respect to their binding activity towards crystalline α-chitin and colloidal chitin, chitin binding assays demonstrated a considerable decrease for mutants W70A and Y245W, and a notable increase for S33W and W231A. When the specific hydrolyzing activity was determined, mutant W231A displayed reduced hydrolytic activity, whilst Y245W showed enhanced activity. This suggested that an alteration in the hydrolytic activity was not correlated with a change in the ability of the enzyme to bind to chitin polymer. A mutation of Trp70 to Ala caused the most severe loss in both the binding and hydrolytic activities, which suggested that it is essential for crystalline chitin binding and hydrolysis. Mutations varied neither the specific hydrolyzing activity against pNP-[GlcNAc]₂, nor the catalytic efficiency against chitohexaose, implying that the mutated residues are not important in oligosaccharide hydrolysis.

Conclusion

Our data provide direct evidence that the binding as well as hydrolytic activities of V. carchariae chitinase A to insoluble chitin are greatly influenced by Trp70 and less influenced by Ser33. Though Trp231 and Tyr245 are involved in chitin hydrolysis, they do not play a major role in the binding process of crystalline chitin and the guidance of the chitin chain into the substrate binding cleft of the enzyme.     

Structural Elucidation of the Bispecificity of A Domains as a Basis for Activating Non‐natural Amino Acids

Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non‐ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered A domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A₁) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the A domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created A domains that were monospecific, or changed the substrate specificity to tryptophan. The non‐natural amino acid 4‐azidophenylalanine is also efficiently activated by a mutant A domain, thus enabling the production of diversified non‐ribosomal peptides for bioorthogonal labeling.     

Analysis of cyathane-type diterpenoids from Cyathus striatus and Hericium erinaceus by high-resolution MALDI MS imaging

Fungal secondary metabolites in both fruiting bodies and pellets from submerged cultures of basidiomycetes were analyzed by atmospheric pressure matrix-assisted laser desorption/ionization-mass spectrometry imaging at a lateral resolution of 15 μm, a mass resolution of 140,000 at m/z 200 and a mass accuracy of better than 2 ppm. The striatals A, B, C, and D, and a number of erinacine type metabolites were detected in the basidiomycetes Cyathus striatus and Hericium erinaceus, respectively. The two fungi were selected as model species, as they are well-known for efficient production of terpenoid secondary metabolites with interesting biological activities, e.g., antibacterial, fungicidal, cytotoxic properties, and stimulating effects on nerve growth factor synthesis. The localization of metabolites revealed a mostly homogeneous distribution of the striatals in the pellets of C. striatus, while a concentration gradient, increasing to the center, was observed in the pellets of H. erinaceus. A mostly homogeneous distribution of metabolites was also found in the fruiting body of H. erinaceus.