Cis-(+)-12-oxophytodienoic acid (OPDA) is a bioactive oxylipin and phytohormone participating in regulation of plant stress responses, growth, and development. Due to its α, β-unsaturated carbonyl moiety, OPDA covalently binds to free thiol groups by Michael addition. This binding, termed OPDAylation, alters the activity of target proteins, such as cyclophilin 20-3 (EC:5.2.1.8) and thioredoxins, that are essential components of the cellular redox regulatory network. To function as a reversible redox regulatory mechanism, OPDAylation should be complemented by a process of de-OPDAylation allowing for fine-tuning of OPDA-dependent regulation. This study explored OPDAylation and de-OPDAylation in vitro with emphasis on the involvement of glutathione. OPDA can be transferred from protein to glutathione (GSH) and vice versa. In a competition experiment, OPDAylation of thioredoxins (TRX) occurred rapidly in the presence of GSH, while over extended incubation times, de-OPDAylation of TRX occurred due to the stoichiometric excess of GSH. These results support the hypothesis that the initial TRX-based OPDAylation is proceeding under kinetic control due to the higher reactivity of the more nucleophilic cysteine moiety in TRX compared to the one of GSH, while the OPDAylation of GSH observed at prolonged incubation time is then the result of a thermodynamically controlled process. De-OPDAylation depends on the protein's sensitivity towards OPDA, the pH and the concentration of excess thiol groups. This likely allows for precise modulation of OPDA amounts, as the rapid modification of protein activity enables subsequent induction of OPDA signaling, whereas de-OPDAylation, triggered by increasing glutathione, increasing cellular reduction or presumably enzymatically, reverses this effect.

Two-component flavin-dependent monooxygenases are of great interest as biocatalysts for the production of pharmaceuticals and other relevant molecules, as they catalyze chemically important reactions such as hydroxylation, epoxidation and halogenation. The monooxygenase components require a separate flavin reductase, which provides the necessary reduced flavin cofactor. The tryptophan halogenase Thal from Streptomyces albogriseolus is a well-characterized two-component flavin-dependent halogenase. Thal exhibits some limitations in terms of halogenation efficiency, also caused by unproductive enzyme-substrate complexes with reduced flavin adenine dinucleotide (FAD). Since the reductase components have an important regulatory function for the activity and efficiency of the monooxygenase by controlling the supply of reduced flavin, here we studied the so far uncharacterized flavin reductase ThdF from the same gene cluster in S. albogriseolus, which potentially cooperates with Thal. A crystal structure of ThdF in complex with both substrates, FAD and NADH, revealed their orientation for hydride transfer. We obtained two further ThdF structures with two FAD molecules bound to the active site, suggesting a ping-pong bi-bi mechanism. In contrast, steady-state enzyme kinetics clearly showed that ThdF catalyzes flavin reduction via an ordered sequential mechanism, with FAD being bound first and FADH2 released last. Compared to related flavin reductases, ThdF has a low kcat and low KM value. The inhibition of ThdF by NAD+ might limit Thal’s halogenation activity when the cellular NADH level is low. These results provide first insights into how the efficiency of Thal could be controlled by flavin reduction at the reductase ThdF.

l ‐Amino acid oxidases (LAAOs) catalyze the oxidative deamination of l ‐amino acids to α‐keto acids. Recombinant production of LAAOs with broad substrate spectrum remains a formidable challenge. We previously achieved this for the highly active and thermostable LAAO4 of Hebeloma cylindrosporum ( Hc LAAO4). Here, we crystallized a proteolytically truncated surface entropy reduction variant of Hc LAAO4 and solved its structure in substrate‐free form and in complex with diverse substrates. The ability to support the aliphatic portion of a substrate's side chain by an overall hydrophobic active site is responsible for the broad substrate spectrum of Hc LAAO4, including l ‐amino acids with big aromatic, acidic and basic side chains. Based on the structural findings, we generated an E288H variant with increased activity toward pharmaceutical building blocks of high interest.

cis-(+)-12-Oxophytodienoic acid (OPDA) is a reactive oxylipin produced by catalytic oxygenation of polyunsaturated α-linolenic acid (18:3 (ω − 3)) in the chloroplast. Apart from its function as precursor for jasmonic acid synthesis, OPDA serves as a signaling molecule and regulator on its own, namely by tuning enzyme activities and altering expression of OPDA-responsive genes. A possible reaction mechanism is the covalent binding of OPDA to thiols via the addition to the C=C double bond of its α,β-unsaturated carbonyl group in the cyclopentenone ring. The reactivity allows for covalent modification of accessible cysteinyl thiols in proteins. This work investigated the reaction of OPDA with selected chloroplast and cytosolic thioredoxins (TRX) and glutaredoxins (GRX) of Arabidopsis thaliana. OPDA reacted with TRX and GRX as detected by decreased m-PEG maleimide binding, consumption of OPDA, reduced ability for insulin reduction and inability to activate glyceraldehyde-3-phosphate dehydrogenase and regenerate glutathione peroxidase (GPXL8), and with lower efficiency, peroxiredoxin IIB (PRXIIB). OPDAylation of certain protein thiols occurs quickly and efficiently in vitro and is a potent post-translational modification in a stressful environment.

Recently, the program INTMSAlign_HiSol for identifying aggregation hotspots in proteins only requiring secondary structure data was introduced. We explored the utility of this program further and applied it for engineering of the aldoxime dehydratase from Bacillus sp. OxB-1. Towards this end, the effect of inverting the hydropathy at selected positions of the amino acid sequence on the enzymatic activity was studied leading to 60% of our constructed variants, which showed improved activity. In part, this activity increase can be rationalised by an improved heme incorporation of the variants. For example, a single mutation gave a 1.8 fold increased enzymatic activity and 30% improved absolute heme incorporation.

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (Vmax app) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s−1·mg protein−1), with a Km value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg2+, Ca2+, Cu2+ and Cd2+, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.

Background Mucopolysaccharidoses (MPS) are monogenic metabolic disorders that significantly affect the skeleton. Eleven enzyme defects in the lysosomal degradation of glycosaminoglycans (GAGs) have been assigned to the known MPS subtypes (I–IX). Arylsulfatase K (ARSK) is a recently characterised lysosomal hydrolase involved in GAG degradation that removes the 2-O-sulfate group from 2-sulfoglucuronate. Knockout of Arsk in mice was consistent with mild storage pathology, but no human phenotype has yet been described. Methods In this study, we report four affected individuals of two unrelated consanguineous families with homozygous variants c.250C>T, p.(Arg84Cys) and c.560T>A, p.(Leu187Ter) in ARSK, respectively. Functional consequences of the two ARSK variants were assessed by mutation-specific ARSK constructs derived by site-directed mutagenesis, which were ectopically expressed in HT1080 cells. Urinary GAG excretion was analysed by dimethylene blue and electrophoresis, as well as liquid chromatography/mass spectrometry (LC-MS)/MS analysis. Results The phenotypes of the affected individuals include MPS features, such as short stature, coarse facial features and dysostosis multiplex. Reverse phenotyping in two of the four individuals revealed additional cardiac and ophthalmological abnormalities. Mild elevation of dermatan sulfate was detected in the two subjects investigated by LC-MS/MS. Human HT1080 cells expressing the ARSK-Leu187Ter construct exhibited absent protein levels by western blot, and cells with the ARSK-Arg84Cys construct showed markedly reduced enzyme activity in an ARSK-specific enzymatic assay against 2-O-sulfoglucuronate-containing disaccharides as analysed by C18-reversed-phase chromatography followed by MS. Conclusion Our work provides a detailed clinical and molecular characterisation of a novel subtype of mucopolysaccharidosis, which we suggest to designate subtype X.

Coenzyme Q 10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para -hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli . CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.

Mucopolysaccharidoses comprise a group of rare metabolic diseases, in which the lysosomal degradation of glycosaminoglycans (GAGs) is impaired due to genetically inherited defects of lysosomal enzymes involved in GAG catabolism. The resulting intralysosomal accumulation of GAG-derived metabolites consequently manifests in neurological symptoms and also peripheral abnormalities in various tissues like liver, kidney, spleen and bone. As each GAG consists of differently sulfated disaccharide units, it needs a specific, but also partly overlapping set of lysosomal enzymes to accomplish their complete degradation. Recently, we identified and characterized the lysosomal enzyme arylsulfatase K (Arsk) exhibiting glucuronate-2-sulfatase activity as needed for the degradation of heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS). In the present study, we investigated the physiological relevance of Arsk by means of a constitutive Arsk knockout mouse model. A complete lack of glucuronate desulfation was demonstrated by a specific enzyme activity assay. Arsk-deficient mice show, in an organ-specific manner, a moderate accumulation of HS and CS metabolites characterized by 2-O-sulfated glucuronate moieties at their non-reducing ends. Pathophysiological studies reflect a rather mild phenotype including behavioral changes. Interestingly, no prominent lysosomal storage pathology like bone abnormalities were detected. Our results from the Arsk mouse model suggest a new although mild form of mucopolysacharidose (MPS), which we designate MPS type IIB.

Ion mobility spectrometry (IMS) coupled with mass spectrometry (MS) enables the investigation of protein folding in solution. Herein, a proof-of-concept for obtaining structural information about the folding of a protein in dependency of the amount of an organic cosolvent in the aqueous medium by means of this IMS-MS method is presented. By analyzing the protein with native nano-electrospray ionization IMS-MS, the impact of acetonitrile as a representative organic cosolvent and/or pH values on the folding of an enzyme was successfully evaluated in a fast and straightforward fashion, as exemplified for an ene reductase from Gluconobacter oxydans. The IMS-MS results are in agreement with findings from the nicotinamide adenine dinucleotide phosphate (NADPH)-based spectrophotometric enzyme activity tests under analogous conditions, and thus, also rationalizing these "wet" analytical data. For this ene reductase, a higher tolerance against CH3 CN in the presence of a buffer was observed by both analytical methods. The results suggest that this IMS-MS methodology could be a useful complementary tool to existing methods in process optimization and fine-tuning of solvent conditions for biotransformations.

Halogenases are valuable biocatalysts for selective C-H activation, but despite recent efforts to broaden their application scope by means of protein engineering, improvement of thermostability and catalytic efficiency is still desired. A directed evolution campaign aimed at generating a thermostable flavin-dependent tryptophan 6-halogenase with reasonable activity suitable for chemoenzymatic purposes. These characteristics were tackled by combining successive rounds of epPCR along with semi-rational mutagenesis leading to a triple mutant (Thal-GLV) with substantially increased thermostability (T-M=23.5 K) and higher activity at 25 degrees C than the wild type enzyme. Moreover, an active-site mutation has a striking impact on thermostability but also on enantioselectivity. Our data contribute to a detailed understanding of biohalogenation and provide a profound basis for future engineering strategies to facilitate chemoenzymatic application of these attractive biocatalysts.

The first calcium complex from nature, Coumamarin (1), 7-hydroxy-3-methoxy-2-oxo-2H-chromene-6-carboxylate Ca(II) complex, was isolated from Aspergillus sydowii ASTI, together with diorcinol (2), violaceol I (3), hydroxysydonic acid (4), cyclo (Trp-Phe), kojic acid, ergosterol, and uracil. The producing strain was isolated from marine water sample collected from Tiran Island, Red Sea, Egypt. Structure 1 was assigned by intensive 1D, 2D NMR, HR-ESIMS, and X-ray crystallography as well. Coumamarin is potentially active against certain tested bacteria and yeasts, while showing no cytotoxic activity against human cervix carcinoma cell line (KB-3-1). Taxonomically, the fungus was identified by phylogenetic analysis of its 18S rRNA gene sequence.

Today, bottom-up protein identification in MALDI-MS is based on employing singly charged peptide ions, which are predominantly formed in the ionization process. However, peptide mass fingerprinting (PMF) with subsequent tandem MS confirmation using these peptide ions is often hampered due to the lower quality of fragment ion mass spectra caused by the higher collision energy necessary for fragmenting singly protonated peptides. Accordingly, peptide ions of higher charge states would be of high interest for analytical purposes, but they are usually not detected in MALDI-MS experiments as they overlap with singly charged matrix clusters and peptide ions. However, when utilizing ion mobility spectrometry (IMS), doubly charged peptide ions can be actively used by separating them from the singly protonated peptides, visualized, and selectively targeted for tandem MS experiments. The generated peptide fragment ion spectra can be used for a more confident protein identification using PMF with tandem MS confirmation, as most doubly protonated peptide ions yield fragment ion mass spectra of higher quality compared to tandem mass spectra of the corresponding singly protonated precursor ions. Mascot protein scores can be increased by approximately 50% when using tandem mass spectra of doubly charged peptide ions, with ion scores up to six times higher compared with ion scores of tandem mass spectra from singly charged precursors.

Arabidopsis (Arabidopsis thaliana) 12-oxophytodienoic acid reductase isoform 3 (OPR3) is involved in the synthesis of jasmonic acid (JA) by reducing the α,β-unsaturated double bond of the cyclopentenone moiety in 12-oxophytodienoic acid (12-OPDA). Recent research revealed that JA synthesis is not strictly dependent on the peroxisomal OPR3. The ability of OPR3 to reduce trinitrotoluene suggests that the old yellow enzyme homolog OPR3 has additional functions. Here, we show that OPR3 catalyzes the reduction of a wide spectrum of electrophilic species that share a reactivity toward the major redox buffers glutathione (GSH) and ascorbate (ASC). Furthermore, we show that 12-OPDA reacts with ASC to form an ASC-12-OPDA adduct, but in addition OPR3 has the ability to regenerate ASC from monodehydroascorbate. The presented data characterize OPR3 as a bifunctional enzyme with NADPH-dependent α,β-ketoalkene double-bond reductase and monodehydroascorbate reductase activities (MDHAR). opr3 mutants showed a slightly less-reduced ASC pool in leaves in line with the MDHAR activity of OPR3 in vitro. These functions link redox homeostasis as mediated by ASC and GSH with OPR3 activity and metabolism of reactive electrophilic species.

Enzymatic digestion of proteins is one of the key steps in proteomic analyses. There has been a steady progress in the applied digestion protocols in the past, starting from conventional time-consuming in-solution or in-gel digestion protocols to rapid and efficient methods utilizing different types of microscale enzyme reactors. Application of such microreactors has been proven beneficial due to lower sample consumption, higher sensitivity and straightforward coupling with LC-MS set-ups. Novel stationary phases, immobilization techniques and device formats are being constantly developed and tested to optimize digestion efficiency of proteolytic enzymes. This review focuses on the latest developments associated with the preparation and application of microscale enzyme reactors for proteomics applications since 2008 onwards. A special attention has been paid to the discussion of different stationary phases applied for immobilization purposes. (C) 2013 Elsevier B.V. All rights reserved.

The successive loss of two benzyl radicals from the [M +Na](+) ions of the isomeric dihydroxybenzene dibenzyl ethers (2-4) and of the isomeric trihydroxybenzene tribenzyl ethers (5-7) under ESI/CID conditions has been studied by deuterium labelling, MS experiments and DFT calculations. The fragmentation of the [M + Na](+) ions of 2 and 4 consists exclusively of the consecutive losses of two C7H7* (benzyl) radicals. This process is largely suppressed in the corresponding [M + Na](+) ions of the meta isomer 3 and also in those of the 1,3,5-analog, phloroglucinol tribenzyl ether (6), suggesting the facile formation of sodiated ortho- and para-quinone ions, [C6H4O2 + Na](+), in the cases of 2 and 4, respectively. This finding is corroborated by a detailed investigation of the sodiated tribenzyl ethers of pyrogallol, [5 + Na](+), and 1,2,4-trihydroxybenzene, [7 + Na](+), and their isotopologs bearing differently labelled benzyl residues. Again, the successive loss of two C7H7* radicals is the only fragmentation channel for ions [7 + Na](+) and strongly predominates for ions [5 + Na](+), with the primary and secondary losses being highly regiospecific: ions [5 + Na](+) lose the first benzyl residue almost exclusively from the central position (0-2) and only then a lateral C7H7* radical (from 0-1 or 0-3). Surprisingly, the very minor primary loss of a lateral benzyl group is followed by that of the other lateral one, suggesting a two-step isomerization process initiated by a 1,4-H* shift. Ions [7 + Na](+) lose the first benzyl radical almost exclusively from 0-1, in very minor amounts from 0-2 but not at all from 0-4. The secondary loss of C7H7* subsequent to the major primary loss occurs from both 0-2 and 0-4 in similar amounts, reflecting the relative stabilities of the sodiated benzyloxy-substituted ortho- and para-benzoquinones. This and several other details of the energy profiles associated with the twofold benzyl loss from ions [5 + Na](+) and [7 + Na](+) were calculated by use of the DFT methodology and were found to agree very well with the regioselectivities observed. Finally, the pyrogallol-based ions [5 + Na](+) were found to eliminate benzaldehyde in minor but significant relative amounts, which takes place with high regioselectivity from 0-1 or 0-3. (C) 2014 Elsevier B.V. All rights reserved.

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e. g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.

In this protocol, a strategy is described for preparing affinity media with monolithic materials as stationary phase, which is exemplified for the biotin-avidin interaction pair. The capillary columns prepared in this manner are compatible with nano-liquid chromatographic conditions. Our protocol is easily adapted to the preparation of specific affinity media with different functionalities and as such provides a platform for a multitude of applications.