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1.
How superoxide reductases and flavodiiron proteins combat oxidative stress in anaerobes.
Martins, MC, Romão, CV, Folgosa, F, Borges, PT, Frazão, C, Teixeira, M
Free radical biology & medicine. 2019;:36-60
Abstract
Microbial anaerobes are exposed in the natural environment and in their hosts, even if transiently, to fluctuating concentrations of oxygen and its derived reactive species, which pose a considerable threat to their anoxygenic lifestyle. To counteract these stressful conditions, they contain a multifaceted array of detoxifying systems that, in conjugation with cellular repairing mechanisms and in close crosstalk with metal homeostasis, allow them to survive in the presence of O2 and reactive oxygen species. Some of these systems are shared with aerobes, but two families of enzymes emerged more recently that, although not restricted to anaerobes, are predominant in anaerobic microbes. These are the iron-containing superoxide reductases, and the flavodiiron proteins, endowed with O2 and/or NO reductase activities, which are the subject of this Review. A detailed account of their physicochemical, physiological and molecular mechanisms will be presented, highlighting their unique properties in allowing survival of anaerobes in oxidative stress conditions, and comparing their properties with the most well-known detoxifying systems.
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2.
Comparative review of the recent enzymatic methods used for selective assay of l-lysine.
Isobe, K, Matsui, D, Asano, Y
Analytical biochemistry. 2019;:113335
Abstract
l-Lysine is an essential amino acid important for maintaining human health. To date, many enzymatic methods for assay of l-lysine have been developed. The first method has been developed using l-lysine α-oxidase (l-LysOα). However, low specificity towards l-lysine of l-LysOα is a disadvantage inherent in this method. Recently, methods more specific to l-lysine were developed using newly discovered enzymes such as l-lysine ε-oxidase (l-LysOε), l-amino acid oxidase/monooxygenase (l-AAO/MOG) and l-lysine decarboxylase/oxidase (l-Lys-DC/OD). The present paper reviews recent enzymatic methods used for assay of l-lysine. These l-lysine selective assays rely on detecting and quantifying hydrogen peroxide, a product generated by the oxidase reaction of these enzymes. l-LysOε catalyzes the oxidative deamination of the ε-amino group of l-lysine, thus assays using this enzyme are more specific towards l-lysine than the ones using l-LysOα. The l-AAO/MOG has high substrate specificity towards l-lysine; however it exhibits l-lysine oxidase and monooxygenase activities. The sensitivity of l-AAO/MOG method was improved either by using its mutant, which has reduced monooxygenase activity, or by coupling with an aminoamide-oxidizing enzyme. The l-Lys-DC/OD exhibits both l-lysine decarboxylase and oxidase activities. The sensitivity of the l-Lys-DC/OD method was improved by using putrescine oxidase to oxidize the decarboxylation product of l-lysine.
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3.
Identification of 4-methyl-5-oxo-octane-1,8-dioic acid and the derivatives as metabolites of steroidal C,D-ring degradation in Comamonas testosteroni TA441.
Horinouchi, M, Malon, M, Hirota, H, Hayashi, T
The Journal of steroid biochemistry and molecular biology. 2019;:277-286
Abstract
Comamonas testosteroni TA441 degrades steroids via 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, which is presumed to be further degraded by β-oxidation. In the β-oxidation process, Coenzyme A (CoA)-ester of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid is produced and converted by β-ketoacyl-CoA-transferase encoded by ORF1 and ORF2 (scdL1L2) to cleave the remaining C-ring. In this study, we isolated and identified 4-methyl-5-oxo-octane-1,8-dioic acid and 4-methyl-5-oxo-3-octene-1,8-dioic acid from the culture of the ORF3 (scdN)-null mutant as metabolites of steroid degradation (ADD and cholic acid analogues; cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid). In addition of these compounds, UHPLC/MS analysis of the culture of the scdN-null mutant revealed significant accumulation of another compound, which was detected as a dominant peak of m/z 155 ([M-CO2]-) accompanied by a small peak of parental ion (m/z 199 [M-]). On the bases of experimental data, this compound was presumed to be 4-methyl-5-oxo-2-octene-1,8-dioic acid, whose CoA-ester was indicated to be converted by scdN-encoded CoA-hydratase into the CoA-ester of 3-hydroxy-4-methyl-5-oxooctan-1,7-carboxylic acid.
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4.
Investigation of quaternary structure of aggregating 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans: In the pursuit of consensus of various biophysical techniques.
Sofińska, K, Wojtkiewicz, AM, Wójcik, P, Zastawny, O, Guzik, M, Winiarska, A, Waligórski, P, Cieśla, M, Barbasz, J, Szaleniec, M
Biochimica et biophysica acta. General subjects. 2019;(6):1027-1039
Abstract
In this work we analyzed the quaternary structure of FAD-dependent 3-ketosteroid dehydrogenase (AcmB) from Sterolibacterium denitrificans, the protein that in solution forms massive aggregates (>600 kDa). Using size-excursion chromatography (SEC), dynamic light scattering (DLS), native-PAGE and atomic force microscopy (AFM) we studied the nature of enzyme aggregation. Partial protein de-aggregation was facilitated by the presence of non-ionic detergent such as Tween 20 or by a high degree of protein dilution but not by addition of a reducing agent or an increase of ionic strength. De-aggregating influence of Tween 20 had no impact on either enzyme's specific activity or FAD reconstitution to recombinant AcmB. The joint experimental (DLS, isoelectric focusing) and theoretical investigations demonstrated gradual shift of enzyme's isoelectric point upon aggregation from 8.6 for a monomeric form to even 5.0. The AFM imaging on mica or highly oriented pyrolytic graphite (HOPG) surface enabled observation of individual protein monomers deposited from a highly diluted solution (0.2 μg/ml). Such approach revealed that native AcmB can indeed be monomeric. AFM imaging supported by theoretical random sequential adsorption (RSA) kinetics allowed estimation of distribution enzyme forms in the bulk solution: 5%, monomer, 11.4% dimer and 12% trimer. Finally, based on results of AFM as well as analysis of the surface of AcmB homology models we have observed that aggregation is most probably initiated by hydrophobic forces and then assisted by electrostatic attraction between negatively charged aggregates and positively charged monomers.
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5.
Enzymatic radiosynthesis of a 18F-Glu-Ureido-Lys ligand for the prostate-specific membrane antigen (PSMA).
Lowe, PT, Dall'Angelo, S, Fleming, IN, Piras, M, Zanda, M, O'Hagan, D
Organic & biomolecular chemistry. 2019;(6):1480-1486
Abstract
Prostate cancer represents a major public health threat as it is one of the most common male cancers worldwide. The prostate-specific membrane antigen (PSMA) is highly over-expressed in prostatic cancer cells in a manner that correlates with both tumour stage and clinical outcome. As such, PSMA has been identified as an attractive target for both imaging and treatment of prostate cancer. In recent years the focus on urea-based peptidomimetic inhibitors of the PSMA (representing low molecular weight/high affinity binders) has intensified as they have found use in the clinical imaging of prostate tumours. Reported herein are the design, synthesis and evaluation of a new fluorinated PSMA targeting small-molecule, FDA-PEG-GUL, which possesses the Glu-NH-CO-NH-Lys pharmacophore conjugated to a 5'-fluorodeoxy-adenosine unit. Inhibition assays were performed with FDA-PEG-GUL which revealed that it inhibits the PSMA in the nanomolar range. Additionally, it has been purposely designed so that it can be produced using the fluorinase enzyme from its chlorinated precursor, allowing for the enzymatic synthesis of radiolabelled [18F]FDA-PEG-GUL via a nucleophilic reaction that takes place in experimentally advantageous conditions (in water at neutral pH and at ambient temperature). Specific binding of [18F]FDA-PEG-GUL to PSMA expressing cancer cells was demonstrated, validating it as a promising PSMA diagnostic tool. This work establishes a successful substrate scope expansion for the fluorinase and demonstrates its first application towards targeting the PSMA.
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6.
Fungal PQQ-dependent dehydrogenases and their potential in biocatalysis.
Takeda, K, Umezawa, K, Várnai, A, Eijsink, VG, Igarashi, K, Yoshida, M, Nakamura, N
Current opinion in chemical biology. 2019;:113-121
Abstract
In 2014, the first fungal pyrroloquinoline-quinone (PQQ)-dependent enzyme was discovered as a pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH). This discovery laid the foundation for a new Auxiliary Activities (AA) family, AA12, in the Carbohydrate-Active enZymes (CAZy) database and revealed a novel enzymatic activity potentially involved in biomass conversion. This review summarizes recent progress made in research on this fungal oxidoreductase and related enzymes. CcPDH consists of the catalytic PQQ-binding AA12 domain, an N-terminal cytochrome b AA8 domain, and a C-terminal family 1 carbohydrate-binding module (CBM1). CcPDH oxidizes 2-keto-d-glucose (d-glucosone), l-fucose, and rare sugars such as d-arabinose and l-galactose, and can activate lytic polysaccharide monooxygenases (LPMOs). Bioinformatic studies suggest a widespread occurrence of quinoproteins in eukaryotes as well as prokaryotes.
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7.
Multicopper oxidases: Biocatalysts in microbial pathogenesis and stress management.
Kaur, K, Sharma, A, Capalash, N, Sharma, P
Microbiological research. 2019;:1-13
Abstract
The acquisition of metal ions such as iron, copper and manganese is essential for the survival of microorganisms as these are constituents of metalloproteins including enzymes, storage proteins, structural elements, transcription factors and antimicrobial factors in various biological processes. However, excess of these metal ions is associated with significant toxicity due to spontaneous redox cycling of ions and obstruction of normal metabolic pathways. To overcome this, microbes have developed a variety of metal regulatory systems allowing them to adapt to the changing biotic and abiotic environments. Multi-copper oxidases (MCOs) such as ceruloplasmins, ferroxidases, laccases and nitrite reductases are such regulatory systems employed by microbes to resist the toxicity of metal ions by controlling their oxidation states under aerobic conditions. MCOs help pathogens survive during an infection by evasion of the toxic environment generated by the host immune system and thus are considered necessary determinants of virulence. This review summarizes the role of MCOs in metal homeostasis under stressful conditions and the extent to which these MCOs contribute to microbial virulence within the host that might prove as an esteemed avenue for the development of novel antimicrobial therapies.
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8.
Alternative oxidase is an important player in the regulation of nitric oxide levels under normoxic and hypoxic conditions in plants.
Kumari, A, Pathak, PK, Bulle, M, Igamberdiev, AU, Gupta, KJ
Journal of experimental botany. 2019;(17):4345-4354
Abstract
Plant mitochondria possess two different pathways for electron transport from ubiquinol: the cytochrome pathway and the alternative oxidase (AOX) pathway. The AOX pathway plays an important role in stress tolerance and is induced by various metabolites and signals. Previously, several lines of evidence indicated that the AOX pathway prevents overproduction of superoxide and other reactive oxygen species. More recent evidence suggests that AOX also plays a role in regulation of nitric oxide (NO) production and signalling. The AOX pathway is induced under low phosphate, hypoxia, pathogen infections, and elicitor treatments. The induction of AOX under aerobic conditions in response to various stresses can reduce electron transfer through complexes III and IV and thus prevents the leakage of electrons to nitrite and the subsequent accumulation of NO. Excess NO under various stresses can inhibit complex IV; thus, the AOX pathway minimizes nitrite-dependent NO synthesis that would arise from enhanced electron leakage in the cytochrome pathway. By preventing NO generation, AOX can reduce peroxynitrite formation and tyrosine nitration. In contrast to its function under normoxia, AOX has a specific role under hypoxia, where AOX can facilitate nitrite-dependent NO production. This reaction drives the phytoglobin-NO cycle to increase energy efficiency under hypoxia.
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9.
MnO2 nanosheets as the biomimetic oxidase for rapid and sensitive oxalate detection combining with bionic E-eye.
Gan, Y, Hu, N, He, C, Zhou, S, Tu, J, Liang, T, Pan, Y, Kirsanov, D, Legin, A, Wan, H, et al
Biosensors & bioelectronics. 2019;:254-261
Abstract
Urolithiasis commonly occurs in kidney and ureteral, and may cause local organ/tissue damage, even kidney failure. The incidence of this disease is increasing worldwide, in which calcium oxalate is the major composition forming the urinary calculus. Therefore, to monitor this disease for the prevention and treatment, measuring the oxalate in the urine is of great significance. Here, a rapid and sensitive colorimetric method was developed based on 3,3',5,5'-tetramethylbenzidine-manganese dioxide (TMB-MnO2) nanosheets for oxalate detection. MnO2 nanosheets acted as an efficient biomimetic oxidase to catalyze the reaction with TMB and oxalate. Pale yellow TMB can be oxidized to blue oxide TMB catalyzed by BSA-stabilized MnO2 nanosheets, and oxalate can selectively inhibit this reaction by consuming and reacting with MnO2 nanosheets, thus achieving the quantitative detection of oxalate. Moreover, a home-made bionic electronic-eye (E-eye) system was developed as a portable in-situ detection platform to efficiently measure the oxalate concentrations in 10 s by direct photographing. By optimizing experimental conditions, this method shows a wide linear range (7.8 μM to 250 μM) and a low detection limit (0.91 μM) for oxalate detection. Besides, this method exhibits high selectivity even with 80-fold interfering chemicals. Furthermore, the performance of the method was validated by testing the artificial urine samples, indicating its great potential for monitoring and diagnosis of urolithiasis in point-of-care applications.
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10.
Functional interactions between nitrite reductase and nitric oxide reductase from Paracoccus denitrificans.
Albertsson, I, Sjöholm, J, Ter Beek, J, Watmough, NJ, Widengren, J, Ädelroth, P
Scientific reports. 2019;(1):17234
Abstract
Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd1 nitrite reductase (cd1NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd1NiR, presumably because cd1NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd1NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd1NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd1NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.