Co-option of an extracellular protease for transcriptional control of nutrient degradation in the fungus Aspergillus nidulans.

Nutrient acquisition is essential for all organisms. Fungi regulate their metabolism according to environmental nutrient availability through elaborate transcription regulatory programs. In filamentous fungi, a highly conserved GATA transcription factor AreA and its co-repressor NmrA govern expression of genes involved in extracellular breakdown, uptake, and metabolism of nitrogen nutrients. Here, we show that the Aspergillus nidulans PnmB protease is a moonlighting protein with extracellular and intracellular functions for nitrogen acquisition and metabolism. PnmB serves not only as a secreted protease to degrade extracellular nutrients, but also as an intracellular protease to control the turnover of the co-repressor NmrA, accelerating AreA transcriptional activation upon nitrogen starvation. PnmB expression is controlled by AreA, which activates a positive feedback regulatory loop. Hence, we uncover a regulatory mechanism in the well-established controls determining the response to nitrogen starvation, revealing functional evolution of a protease gene for transcriptional regulation and extracellular nutrient breakdown.

Aspergillus nidulans AmyG Functions as an Intracellular α-Amylase to Promote α-Glucan Synthesis.

α-Glucan is a major cell wall component and a virulence and adhesion factor for fungal cells. However, the biosynthetic pathway of α-glucan was still unclear. α-Glucan was shown to be composed mainly of 1,3-glycosidically linked glucose, with trace amounts of 1,4-glycosidically linked glucose. Besides the α-glucan synthetases, amylase-like proteins were also important for α-glucan synthesis. In our previous work, we showed that Aspergillus nidulans AmyG was an intracellular protein and was crucial for the proper formation of α-glucan. In the present study, we expressed and purified AmyG in an Escherichia coli system. Enzymatic characterization found that AmyG mainly functioned as an α-amylase that degraded starch into maltose. AmyG also showed weak glucanotransferase activity. Most intriguingly, supplementation with maltose in shaken liquid medium could restore the α-glucan content and the phenotypic defect of a ΔamyG strain. These data suggested that AmyG functions mainly as an intracellular α-amylase to provide maltose during α-glucan synthesis in A. nidulans. IMPORTANCE Short α-1,4-glucan was suggested as the primer structure for α-glucan synthesis. However, the exact structure and its source remain elusive. AmyG was essential to promote α-glucan synthesis and had a major impact on the structure of α-glucan in the cell wall. Data presented here revealed that AmyG belongs to the GH13_5 family and showed strong amylase function, digesting starch into maltose. Supplementation with maltose efficiently rescued the phenotypic defect and α-glucan deficiency in an ΔamyG strain but not in an ΔagsB strain. These results provide the first piece of evidence for the primer structure of α-glucan in fungal cells, although it might be specific to A. nidulans.

Nitroreductase Increases Menadione-Mediated Oxidative Stress in Aspergillus nidulans.

Nitroreductases (NTRs) catalyze the reduction of a wide range of nitro-compounds and quinones using NAD(P)H. Although the physiological functions of these enzymes remain obscure, a tentative function of resistance to reactive oxygen species (ROS) via the detoxification of menadione has been proposed. This suggestion is based primarily on the transcriptional or translational induction of an NTR response to menadione rather than on convincing experimental evidence. We investigated the performance of a fungal NTR from Aspergillus nidulans (AnNTR) exposed to menadione to address the question of whether NTR is really an ROS defense enzyme. We confirmed that AnNTR was transcriptionally induced by external menadione. We observed that menadione treatment generated cytotoxic levels of O2•-, which requires well-known antioxidant enzymes such as superoxide dismutase, catalase, and peroxiredoxin to protect A. nidulans against menadione-derived ROS stress. However, AnNTR was counterproductive for ROS defense, since knocking out AnNTR decreased the intracellular O2•- levels, resulting in fungal viability higher than that of the wild type. This observation implies that AnNTR may accelerate the generation of O2•- from menadione. Our in vitro experiments indicated that AnNTR uses NADPH to reduce menadione in a single-electron reaction, and the subsequent semiquinone-quinone redox cycling resulted in O2•- generation. We demonstrated that A. nidulans nitroreductase should be an ROS generator, but not an ROS scavenger, in the presence of menadione. Our results clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous. IMPORTANCE Menadione is commonly used as an O2•- generator in studies of oxidative stress responses. However, the precise mechanism through which menadione mediates cellular O2•- generation, as well as the way in which cells respond, remains unclear. Elucidating these events will have important implications for the use of menadione in biological and medical studies. Our results show that the production of Aspergillus nidulans nitroreductase (AnNTR) was induced by menadione. However, the accumulated AnNTR did not protect cells but instead increased the cytotoxic effect of menadione through a single-electron reduction reaction. Our finding that nitroreductase is involved in the menadione-mediated O2•- generation pathway has clarified the relationship between nitroreductase and menadione-derived ROS stress, which has long been ambiguous.

The putative sensor histidine kinase VadJ coordinates development and sterigmatocystin production in Aspergillus nidulans.

The VosA-VelB heterocomplex governs expression of several genes associated with fungal development and secondary metabolism. In this study, we have investigated the functions of one of the VosA-VelB-activated developmental genes vadJ in development and production of the mycotoxin sterigmatocystin in the model fungus Aspergillus nidulans. The vadJ gene is predicted to encode a 957-amino acid length protein containing a highly conserved sensor histidine kinase domain. The deletion of vosA or velB resulted in decreased mRNA levels of vadJ throughout the life cycle, suggesting that VosA and VelB are necessary for proper expression of vadJ. Nullifying vadJ led to highly restricted colony growth, lowered formation of asexual spores, and about two-fold reduction in conidial viability. Conversely, the deletion of vadJ resulted in elevated production of sexual fruiting bodies and sterigmatocystin. These suggest that VadJ is necessary for proper coordination of asexual and sexual development, and sterigmatocystin production. In accordance with this idea, the deletion of vadJ led to elevated mRNA levels of the two key sexual developmental activators esdC and nsdD. In summary, the putative sensor histidine kinase VadJ represses sexual development and sterigmatocystin production, but activates asexual development in A. nidulans.

An Aspergillus nidulans endo-ß-1,3-glucanase exhibited specific catalytic features and was used to prepare 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose with high antioxidant activity from barley ß-glucan and laminarin, respectively.

An endo-ß-1,3(4)-glucanase AnENG16A from Aspergillus nidulans shows distinctive catalytic features for hydrolysis of ß-glucans. AnENG16A hydrolyzed Eisenia bicyclis laminarin to mainly generate 3-O-ß-gentiobiosyl-d-glucose and hydrolyzed barley ß-glucan to mainly produce 3-O-ß-cellobiosyl-d-glucose. Using molecular exclusion chromatography, we isolated and purified 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose, respectively, from AnENG16A-hydrolysate of barley ß-glucan and E. bicyclis laminarin. Further study reveals that 3-O-ß-cellobiosyl-d-glucose had 8.99-fold higher antioxidant activity than barley ß-glucan and 3-O-ß-gentiobiosyl-d-glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin. Notably, 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose exhibited 148.9% and 116.0% higher antioxidant activity than laminaritriose, respectively, indicating that ß-1,4-linkage or -1,6-linkage at non-reducing end of ß-glucotrioses had enhancing effect on antioxidant activity compared to ß-1,3-linkage. Furthermore, 3-O-ß-cellobiosyl-d-glucose showed 237.9% higher antioxidant activity than cellotriose, and laminarin showed 5.06-fold higher antioxidant activity than barley ß-glucan, indicating that ß-1,4-linkage at reducing end of ß-glucans or oligosaccharides resulted in decrease of antioxidant activity compared to ß-1,3-linkage.

Galactofuranose (Galf)-containing sugar chain contributes to the hyphal growth, conidiation and virulence of F. oxysporum f.sp. cucumerinum.

The ascomycete fungus Fusarium oxysporum f.sp. cucumerinum causes vascular wilt diseases in cucumber. However, few genes related to morphogenesis and pathogenicity of this fungal pathogen have been functionally characterized. BLASTp searches of the Aspergillus fumigatus UgmA and galatofuranosyltransferases (Galf-transferases) sequences in the F. oxysporum genome identified two genes encoding putative UDP-galactopyranose mutase (UGM), ugmA and ugmB, and six genes encoding putative Galf-transferase homologs. In this study, the single and double mutants of the ugmA, ugmB and gfsB were obtained. The roles of UGMs and GfsB were investigated by analyzing the phenotypes of the mutants. Our results showed that deletion of the ugmA gene led to a reduced production of galactofuranose-containing sugar chains, reduced growth and impaired conidiation of F. oxysporum f.sp. cucumerinum. Most importantly, the ugmA deletion mutant lost the pathogenicity in cucumber plantlets. Although deletion of the ugmB gene did not cause any visible phenotype, deletion of both ugmA and ugmB genes caused more severe phenotypes as compared with the ΔugmA, suggesting that UgmA and UgmB are redundant and they can both contribute to synthesis of UDP-Galf. Furthermore, the ΔgfsB exhibited an attenuated virulence although no other phenotype was observed. Our results demonstrate that the galactofuranose (Galf) synthesis contributes to the cell wall integrity, germination, hyphal growth, conidiation and virulence in Fusarium oxysporum f.sp. cucumerinum and an ideal target for the development of new anti-Fusarium agents.

The fungal RABOME: RAB GTPases acting in the endocytic and exocytic pathways of Aspergillus nidulans (with excursions to other filamentous fungi).

RAB GTPases are major determinants of membrane identity that have been exploited as highly specific reporters to study intracellular traffic in vivo. A score of fungal papers have considered individual RABs, but systematic, integrated studies on the localization and physiological role of these regulators and their effectors have been performed only with Aspergillus nidulans. These studies have influenced the intracellular trafficking field beyond fungal specialists, leading to findings such as the maturation of trans-Golgi (TGN) cisternae into post-Golgi RAB11 secretory vesicles, the concept that these RAB11 secretory carriers are loaded with three molecular nanomotors, the understanding of the role of endocytic recycling mediated by RAB6 and RAB11 in determining the hyphal mode of life, the discovery that early endosome maturation and the ESCRT pathway are essential, the identification of specific adaptors of dynein-dynactin to RAB5 endosomes, the exquisite dependence that autophagy displays on RAB1 activity, the role of TRAPPII as a GEF for RAB11, or the conclusion that the RAB1-to-RAB11 transition is not mediated by TRAPP maturation. A remarkable finding was that the A. nidulans Spitzenkörper contains four RABs: RAB11, Sec4, RAB6, and RAB1. How these RABs cooperate during exocytosis represents an as yet outstanding question.

Fungal Dioxygenase AsqJ Is Promiscuous and Bimodal: Substrate-Directed Formation of Quinolones versus Quinazolinones.

Previous studies showed that the FeII /α-ketoglutarate dependent dioxygenase AsqJ induces a skeletal rearrangement in viridicatin biosynthesis in Aspergillus nidulans, generating a quinolone scaffold from benzo[1,4]diazepine-2,5-dione substrates. We report that AsqJ catalyzes an additional, entirely different reaction, simply by a change in substituent in the benzodiazepinedione substrate. This new mechanism is established by substrate screening, application of functional probes, and computational analysis. AsqJ excises H2 CO from the heterocyclic ring structure of suitable benzo[1,4]diazepine-2,5-dione substrates to generate quinazolinones. This novel AsqJ catalysis pathway is governed by a single substituent within the complex substrate. This unique substrate-directed reactivity of AsqJ enables the targeted biocatalytic generation of either quinolones or quinazolinones, two alkaloid frameworks of exceptional biomedical relevance.

Carbon Catabolite Repression in Filamentous Fungi Is Regulated by Phosphorylation of the Transcription Factor CreA.

Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C2H2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects.IMPORTANCE In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.

Type II phosphatidylserine decarboxylase is crucial for the growth and morphogenesis of the filamentous fungus Aspergillus nidulans.

Phosphatidylserine decarboxylases (PSDs) catalyze the production of phosphatidylethanolamine (PE) from phosphatidylserine (PS) and are crucial for the maintenance of PE levels in fungi. The PSDs are classified into two types; the type I PSDs are conserved from bacteria to humans, while the type II PSDs exist only in fungi and plants. In yeasts, the deletion of type I PSD-encoding genes causes severe growth retardation. In contrast, the deletion of type II PSD-encoding genes has little or no effect. In this study, we found four genes encoding type II PSD orthologs in the filamentous fungus Aspergillus nidulans; these included psdB, psdC, psdD, and psdE. Deletion of psdB caused severe growth defects on minimal medium and these defects were partially restored by the addition of ethanolamine, choline, PE, or phosphatidylcholine into the medium. The conidiation efficiency of the psdB deletion mutant was dramatically decreased and its conidiophore structures were aberrant. In the psdB deletion mutant, the PE content decreased while the PS content increased. We further showed that PsdB had a major PSD activity. Our findings suggest that the type II PSDs exert important roles in the phospholipid homeostasis, and in the growth and morphogenesis of filamentous fungi.

Function and regulation of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) deubiquitinase module.

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) chromatin modifying complex is a critical regulator of gene expression and is highly conserved across species. Subunits of SAGA arrange into discrete modules with lysine aceyltransferase and deubiquitinase activities housed separately. Mutation of the SAGA deubiquitinase module can lead to substantial biological misfunction and diseases such as cancer, neurodegeneration, and blindness. Here, we review the structure and functions of the SAGA deubiquitinase module and regulatory mechanisms acting to control these.

Identification and characterization of ß-d-galactofuranosidases from Aspergillus nidulans and Aspergillus fumigatus.

Although ß-d-galactofuranosidases (Galf-ases) that hydrolyze ß-d-galactofuranose (Galf)-containing oligosaccharides have been characterized in various organisms, to date no Galf-specific Galf-ase-encoding genes have been reported in Aspergillus fungi. Based on the amino acid sequences of previously identified bacterial Galf-ases, here we found two candidate Galf-specific Galf-ase genes AN2395 (gfgA) and AN3200 (gfgB) in the genome of Aspergillus nidulans. Indeed, recombinant GfgA and GfgB proteins exhibited Galf-specific Galf-ase activity, but no detectable α-l-arabinofuranosidase (Araf-ase) activity. Phylogenetic analysis of GfgA and GfgB orthologs indicated that there are two types of Aspergillus species: those containing one ortholog each for GfgA and GfgB; and those containing only one ortholog in total, among which Aspergillus fumigatus there is a representative with a single ortholog Galf-ase Afu2g14520. Unlike GfgA and GfgB, the recombinant Afu2g14520 protein showed higher Araf-ase activity than Galf-ase activity. An assay of substrate specificity revealed that although GfgA and GfgB are both exo-type Galf-ases and hydrolyze ß-(1,5) and ß-(1,6) linkages, GfgA hydrolyzes ß-(1,6)-linked Galf-oligosaccharide more effectively as compared with GfgB. Collectively, our findings indicate that Galf-ases in Aspergillus species may have a role in cooperatively degrading Galf-containing oligosaccharides depending on environmental conditions.

Biosynthesis of Mycotoxin Fusaric Acid and Application of a PLP-Dependent Enzyme for Chemoenzymatic Synthesis of Substituted l-Pipecolic Acids.

Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified, but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5'-phosphate (PLP)-dependent enzyme (Fub7), and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic α,ß-unsaturated aldehyde. By harnessing the stereoselective C-C bond-forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl-, 5,5-dialkyl-, and 5,5,6-trialkyl-l-pipecolic acids of high diastereomeric ratio.

Characterization of intra- and inter-species hybrid tetramers of pyruvate carboxylase: Biotin and the BCCP domain play a crucial role in determination of the kinetics and thermodynamics of catalysis.

The formation, kinetics and thermodynamic activation parameters of hybrid tetramers of pyruvate carboxylase (PC) formed between wild-type Rhizobium etli pyruvate carboxylase (WTRePC) and mutant forms of this enzyme, as well as between Aspergillus nidulans PC and mutant forms of RePC have been characterized in a previous study. In this current work, we aim to extend the previous study by forming hybrid tetramers between WTRePC or chicken liver PC (CLPC) with single or double mutant RePCs. By forming hybrid tetramers between WTRePC with either K1119A or ΔBCCP RePC, the biotin moiety and BCCP (biotin carboxyl carrier protein) domain appear to play a crucial role in determination of thermodynamic activation parameters, especially the activation entropy, and the order of tetrameric structure. Using E218A:K1119A hybrid tetramers, an alternative pathway of biotin carboxylation occurred only in the absence of acetyl CoA. In this pathway, the biotin of the E218A subunits is carboxylated in the BC domain of the K1119A subunits, since the E218A mutation destroys the catalytic activity of the BC domain. Transfer of the carboxyl group to pyruvate could then occur in the CT domain of either E218A or K1119A. Part of the reduction of activity in hybrid tetramers of WTRePC and double mutant, E218A.K1119A could result from the loss of this pathway. Previously, D1018A mutant RePC homotetramers exhibited a 12-fold increase in the rate constant for catalysis in the absence of acetyl CoA. This was taken to indicate that inter-residue interactions involving D1018 inhibit the interconversion between the symmetrical and asymmetrical forms of the tetramer in the absence of acetyl CoA. The mutation, D1018A, in hybrid tetramers of WTRePC:D1018A.K1119A (D1018A.K1119A is a double mutant form of RePC) had no such effect on the rate constant, suggesting that in hybrid tetramers obligatory oscillation between asymmetrical and symmetrical conformers of the tetramer is not required to drive the catalytic cycle. Finally, K1119A or E218A RePC mutant can form hybrid tetramers with PC subunits from an evolutionarily distant species, chicken, that have stability characteristics that lie between those of the homotetramers of the two enzymes. This work provides insights into the how the PC tetramer functions to perform catalysis and is regulated by acetyl CoA. The ability to form hybrid tetrameric PCs composed of PC subunits from widely varying species that have a mixture of characteristics of the two source enzymes may also provide ways of developing novel PCs for biotechnological purposes.

The High Osmolarity Glycerol Mitogen-Activated Protein Kinase regulates glucose catabolite repression in filamentous fungi.

The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.

Supplementation of Aspergillus glaucus with gfdB gene encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans.

In Aspergillus nidulans, there are two putative glycerol 3-phosphate dehydrogenases encoded by the genes gfdA and gfdB, while the genome of the osmophilic Aspergillus glaucus harbors only the ortholog of the A. nidulans gfdA gene. Our aim was to insert the gfdB gene into the genome of A. glaucus, and we reached this goal with the adaptation of the Agrobacterium tumefaciens-mediated transformation method. We tested the growth of the gfdB-complemented A. glaucus strains on a medium containing 2 mol l-1 sorbitol in the presence of oxidative stress generating agents such as tert-butyl hydroperoxide, H2 O2 , menadione sodium bisulfite, as well as the cell wall integrity stress-inducing agent Congo Red and the heavy metal stress eliciting CdCl2 . The growth of the complemented strains was significantly higher than that of the wild-type strain on media supplemented with these stress generating agents. The A. nidulans ΔgfdB mutant was also examined under the same conditions and resulted in a considerably lower growth than that of the control strain in all stress exposure experiments. Our results shed light on the fact that the gfdB gene from A. nidulans was also involved in the stress responses of the complemented A. glaucus strains supporting our hypothesis on the antioxidant function of GfdB in the Aspergilli. Nevertheless, the osmotolerant nature of A. glaucus could not be explained by the lack of the gfdB gene in A. glaucus, as we hypothesized earlier.

Isocoumarin formation by heterologous gene expression and modification by host enzymes.

Heterologous expression has been proven to be a successful strategy for the identification of metabolites encoded by cryptic/silent genes. Expression of a nonreducing polyketide synthase (NR-PKS) gene from Penicillium crustosum in Aspergillus nidulans led to the accumulation of three isocoumarins 1-3. Feeding experiments revealed that the PKS product 1 can be converted by the host enzymes to its hydroxylated (2) and methylated (3) derivatives. These results provided one additional example that unexpected further modifications of an enzyme product can take place in a heterologous host.

Characterization of gfdB, putatively encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans.

The genome of Aspergillus nidulans accommodates two glycerol 3-phosphate dehydrogenase genes, gfdA and gfdB. Previous studies confirmed that GfdA is involved in the osmotic stress defence of the fungus. In this work, the physiological role of GfdB was characterized via the construction and functional characterization of the gene deletion mutant ΔgfdB. Unexpectedly, ΔgfdB strains showed oxidative stress sensitivity in the presence of a series of well-known oxidants including tert-butyl-hydroperoxide (tBOOH), diamide as well as hydrogen peroxide. Moderate sensitivity of the mutant towards the cell wall stress inducing agent CongoRed was also observed. Hence, both Gfd isoenzymes contributed to the environmental stress defence of the fungus but their functions were stress-type-specific. Furthermore, the specific activities of certain antioxidant enzymes, like catalase and glutathione peroxidase, were lower in ΔgfdB hyphae than those recorded in the control strain. As a consequence, mycelia from ΔgfdB cultures accumulated reactive species at higher levels than the control. On the other hand, the specific glutathione reductase activity was higher in the mutant, most likely to compensate for the elevated intracellular oxidative species concentrations. Nevertheless, the efficient control of reactive species failed in ΔgfdB cultures, which resulted in reduced viability and, concomitantly, early onset of programmed cell death in mutant hyphae. Inactivation of gfdB brought about higher mannitol accumulation in mycelia meanwhile the erythritol production was not disturbed in unstressed cultures. After oxidative stress treatment with tBOOH, only mannitol was detected in both mutant and control mycelia and the accumulation of mannitol even intensified in the ΔgfdB strain.

Overexpression and Biochemical Characterization of an Endo-α-1,4-polygalacturonase from Aspergillus nidulans in Pichia pastoris.

Pectinases have many applications in the industry of food, paper, and textiles, therefore finding novel polygalacturonases is required. Multiple sequence alignment and phylogenetic analysis of AnEPG (an endo-α-1,4-polygalacturonase from Aspergillus nidulans) and other GH 28 endo-polygalacturonases suggested that AnEPG is different from others. AnEPG overexpressed in Pichia pastoris was characterized. AnEPG showed the highest activity at pH 4.0, and exhibited moderate activity over a narrow pH range (pH 2.0-5.0) and superior stability in a wide pH range (pH 2.0-12.0). It displayed the highest activity at 60 °C, and retained >42.2% of maximum activity between 20 and 80 °C. It was stable below 40 °C and lost activity very quickly above 50 °C. Its apparent kinetic parameters against PGA (polygalacturonic acid) were determined, with the Km and kcat values of 8.3 mg/mL and 5640 µmol/min/mg, respectively. Ba2+ and Ni2+ enhanced activity by 12.2% and 9.4%, respectively, while Ca2+, Cu2+, and Mn2+ inhibited activity by 14.8%, 12.8%, and 10.2% separately. Analysis of hydrolysis products by AnEPG proved that AnEPG belongs to an endo-polygalacturonase. Modelled structure of AnEPG by I-TASSER showed structural characteristics of endo-polygalacturonases. This pectinase has great potential to be used in food industry and as feed additives.

Epoxidation Catalyzed by the Nonheme Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, AsqJ: Mechanistic Elucidation of Oxygen Atom Transfer by a Ferryl Intermediate.

Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by nonheme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the nonheme iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy) to electron-withdrawing groups (e.g., trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations, and molecular dynamic simulations collectively revealed the following mechanistic insights: (1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); (2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from nonheme Fe(IV)-oxo synthetic model complexes; (3) The OAT step most likely proceeds through a stepwise process with the initial formation of a C(benzylic)-O bond to generate an Fe-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.