We present a computational case study of X-ray single-particle imaging of hydrated proteins on an example of 2-Nitrogenase-Iron protein covered with water layers of various thickness, using a start-to-end simulation platform and experimental parameters of the SPB/SFX instrument at the European X-ray Free-Electron Laser facility. The simulations identify an optimal thickness of the water layer at which the effective resolution for imaging the hydrated sample becomes significantly higher than for the non-hydrated sample. This effect is lost when the water layer becomes too thick. Even though the detailed results presented pertain to the specific sample studied, the trends which we identify should also hold in a general case. We expect these findings will guide future single-particle imaging experiments using hydrated proteins.
- MeSH
- difrakce rentgenového záření přístrojové vybavení metody MeSH
- elektrony MeSH
- fotony MeSH
- lasery * MeSH
- molekulární zobrazování metody MeSH
- oxidoreduktasy chemie účinky záření MeSH
- rentgenové záření škodlivé účinky MeSH
- simulace molekulární dynamiky * MeSH
- voda chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurosporacrassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.
- MeSH
- celulosa chemie MeSH
- fungální proteiny chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- hmotnostní spektrometrie MeSH
- katalytická doména MeSH
- katalýza MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- kyslík chemie MeSH
- lignin chemie MeSH
- měď chemie MeSH
- Neurospora crassa enzymologie MeSH
- oxidační stres MeSH
- oxidoreduktasy chemie MeSH
- oxygenasy se smíšenou funkcí chemie MeSH
- polysacharidy chemie MeSH
- reaktivní formy kyslíku chemie MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nonenzymatic oxidative processes in living organisms are among the inevitable consequences of respiration and environmental conditions. These oxidative processes can lead to the formation of two stereoisomers (R and S) of methionine sulfoxide, and the redox balance between methionine and methionine sulfoxide in proteins has profound implications on their function. Methionine oxidation can be reverted enzymatically by methionine sulfoxide reductases (Msrs). The two enzyme classes known to fulfill this role are MsrA, reducing the (S)-isomer, and MsrB, reducing the (R)-isomer of methionine sulfoxide. They are strictly stereoselective and conserved throughout the tree of life. Under stress conditions such as stationary phase and nutrient starvation, Escherichia coli upregulates the expression of MsrA but a similar effect has not been described for MsrB, raising the conundrum of which pathway enables reduction of the (R)-isomer of methionine sulfoxide in these conditions. Using the recently developed chiral fluorescent probes Sulfox-1, we show that in stationary phase-stressed E. coli, MsrA does have a stereocomplementary activity reducing the (R)-isomer of methionine sulfoxide. However, this activity is not provided by MsrB as expected, but instead by the DMSO reductase complex DmsABC, widely conserved in bacteria. This finding reveals an unexpected diversity in the metabolic enzymes of redox regulation concerning methionine, which should be taken into account in any antibacterial strategies exploiting oxidative stress. DATABASE: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013610.
- MeSH
- Escherichia coli enzymologie MeSH
- fluorescenční barviva chemie MeSH
- konformace proteinů MeSH
- methionin analogy a deriváty chemie metabolismus MeSH
- methioninsulfoxidreduktasy chemie metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres * MeSH
- oxidoreduktasy chemie metabolismus MeSH
- proteiny obsahující železo a síru chemie metabolismus MeSH
- proteomika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.
- MeSH
- arginin genetika MeSH
- flavinmononukleotid chemie genetika MeSH
- flaviny genetika metabolismus MeSH
- FMN-reduktasa chemie genetika MeSH
- katalytická doména genetika MeSH
- kinetika MeSH
- konformace proteinů * MeSH
- krystalografie rentgenová MeSH
- oxidace-redukce MeSH
- oxidoreduktasy chemie genetika MeSH
- Paracoccus denitrificans chemie enzymologie MeSH
- sekvence aminokyselin genetika MeSH
- superoxidy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal β-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.
- MeSH
- Agrobacterium enzymologie genetika MeSH
- bakteriální geny MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- flavinmononukleotid metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- kvarterní struktura proteinů MeSH
- molekulární modely MeSH
- NADPH-dehydrogenasa chemie genetika metabolismus MeSH
- operon MeSH
- oxidační stres MeSH
- oxidoreduktasy chemie genetika metabolismus MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Degradation of the plant hormone cytokinin is controlled by cytokinin oxidase/dehydrogenase (CKX) enzymes. The molecular and cellular behavior of these proteins is still largely unknown. In this study, we show that CKX1 is a type II single-pass membrane protein that localizes predominantly to the endoplasmic reticulum (ER) in Arabidopsis (Arabidopsis thaliana). This indicates that this CKX isoform is a bona fide ER protein directly controlling the cytokinin, which triggers the signaling from the ER. By using various approaches, we demonstrate that CKX1 forms homodimers and homooligomers in vivo. The amino-terminal part of CKX1 was necessary and sufficient for the protein oligomerization as well as for targeting and retention in the ER. Moreover, we show that protein-protein interaction is largely facilitated by transmembrane helices and depends on a functional GxxxG-like interaction motif. Importantly, mutations rendering CKX1 monomeric interfere with its steady-state localization in the ER and cause a loss of the CKX1 biological activity by increasing its ER-associated degradation. Therefore, our study provides evidence that oligomerization is a crucial parameter regulating CKX1 biological activity and the cytokinin concentration in the ER. The work also lends strong support for the cytokinin signaling from the ER and for the functional relevance of the cytokinin pool in this compartment.
- MeSH
- Arabidopsis metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- membránové proteiny chemie metabolismus MeSH
- multimerizace proteinu * MeSH
- oxidoreduktasy chemie metabolismus MeSH
- proteinové domény MeSH
- proteiny - lokalizační signály MeSH
- proteiny huseníčku chemie metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Human DHRS7 (SDR34C1) is one of insufficiently described enzymes of the short-chain dehydrogenase/reductase superfamily. The members of this superfamily often play an important pato/physiological role in the human body, participating in the metabolism of diverse substrates (e.g. retinoids, steroids, xenobiotics). A systematic approach to the identification of novel, physiological substrates of DHRS7 based on a combination of homology modeling, structure-based virtual screening and experimental evaluation has been used. Three novel substrates of DHRS7 (dihydrotestosterone, benzil and 4,4'-dimetylbenzil) have been described.
Cytokinins are hormones that regulate plant development and their environmental responses. Their levels are mainly controlled by the cytokinin oxidase/dehydrogenase (CKO), which oxidatively cleaves cytokinins using redox-active electron acceptors. CKO belongs to the group of flavoproteins with an 8α-N1-histidyl FAD covalent linkage. Here, we investigated the role of seven active site residues, H105, D169, E288, V378, E381, P427 and L492, in substrate binding and catalysis of the CKO1 from maize (Zea mays, ZmCKO1) combining site-directed mutagenesis with kinetics and X-ray crystallography. We identify E381 as a key residue for enzyme specificity that restricts substrate binding as well as quinone electron acceptor binding. We show that D169 is important for catalysis and that H105 covalently linked to FAD maintains the enzyme's structural integrity, stability and high rates with electron acceptors. The L492A mutation significantly modulates the cleavage of aromatic cytokinins and zeatin isomers. The high resolution X-ray structures of ZmCKO1 and the E381S variant in complex with N6-(2-isopentenyl)adenosine reveal the binding mode of cytokinin ribosides. Those of ZmCKO2 and ZmCKO4a contain a mobile domain, which might contribute to binding of the N9 substituted cytokinins.
- MeSH
- cytokininy metabolismus MeSH
- flavinadenindinukleotid chemie metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- kukuřice setá enzymologie MeSH
- mutageneze cílená MeSH
- oxidoreduktasy chemie genetika metabolismus MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The metabolism of steroids and retinoids has been studied in detail for a long time, as these compounds are involved in a broad spectrum of physiological processes. Many enzymes participating in the conversion of such compounds are members of the short-chain dehydrogenase/reductase (SDR) superfamily. Despite great effort, there still remain a number of poorly characterized SDR proteins. According to various bioinformatics predictions, many of these proteins may play a role in the metabolism of steroids and retinoids. Dehydrogenase/reductase (SDR family) member 7 (DHRS7) is one such protein. In a previous study, we determined DHRS7 to be an integral membrane protein of the endoplasmic reticulum facing the lumen which has shown at least in vitro NADPH-dependent reducing activity toward several eobiotics and xenobiotics bearing a carbonyl moiety. In the present paper pure DHRS7 was used for a more detailed study of both substrate screening and an analysis of kinetics parameters of the physiologically important substrates androstene-3,17-dione, cortisone and all-trans-retinal. Expression patterns of DHRS7 at the mRNA as well as protein level were determined in a panel of various human tissue samples, a procedure that has enabled the first estimation of the possible biological function of this enzyme. DHRS7 is expressed in tissues such as prostate, adrenal glands, liver or intestine, where its activity could be well exploited. Preliminary indications show that DHRS7 exhibits dual substrate specificity recognizing not only steroids but also retinoids as potential substrates and could be important in the metabolism of these signalling molecules.
- MeSH
- androstendion metabolismus MeSH
- cirkulární dichroismus MeSH
- fylogeneze MeSH
- kinetika MeSH
- kortison metabolismus MeSH
- lidé MeSH
- oxidoreduktasy chemie genetika metabolismus MeSH
- regulace genové exprese enzymů MeSH
- retinaldehyd metabolismus MeSH
- steroidy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
