scholarly journals Structural and Kinetic Characterization of Hyperthermophilic NADH-Dependent Persulfide Reductase from Archaeoglobus fulgidus

Archaea ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Sherwin Shabdar ◽  
Bukuru Anaclet ◽  
Ana Garcia Castineiras ◽  
Neyissa Desir ◽  
Nicholas Choe ◽  
...  
Keyword(s):  
Nadh Oxidase ◽  
Reductase Activity ◽  
Sulfur Metabolism ◽  
Archaeoglobus Fulgidus ◽  
Bacterial Enzymes ◽  
Detectable Activity ◽  
Metabolic Role ◽  
Nadh Oxidase Activity

NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H2S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S0 and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting k cat / K m on the order of 104 M-1 s-1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2 ′ -phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S0 or serve in another metabolic role that has yet to be identified.

Diacetyl reductase of Lactobacillus casei

1970 ◽  
Vol 16 (10) ◽  
pp. 947-951 ◽  
Author(s):  
A. L. Branen ◽  
T. W. Keenan
Keyword(s):  
Lactobacillus Casei ◽  
Nadh Oxidase ◽  
Reductase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Cell Extracts ◽  
Alumina Gel ◽  
Nadh Oxidase Activity ◽  
Nadh Oxidoreductase

Diacetyl reductase (diacetyl:reduced nicotinamide adenine dinucleotide (NADH) oxidoreductase, EC. 1.1.1.5) has been isolated from Lactobacillus casei. Cell sonication, ammonium sulfate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography, and alumina gel adsorption were used to obtain the partially purified enzyme. Both NADH oxidase and diacetyl reductase activity were associated with the same fraction at all stages in purification. Growth in media containing added pyruvate resulted in a 10-fold increase in the NADH oxidase activity and a 3-fold increase in the diacetyl reductase activity of crude cell extracts on a protein basis. Purified preparations showed maximal reductase and oxidase activities at pH 4.5 and 5.0, respectively. Lineweaver–Burke plots yielded intersecting lines when NADH and diacetyl concentrations were varied, suggesting a flavin-linked reaction. The absorption spectrum of the purified preparation was characteristic of that of a flavoprotein. The product of the reduction of diacetyl was identified as acetoin. Acetoin and methylene blue were inactive as acceptors.

scholarly journals Identification and Characterization of AIFsh2, a Mitochondrial Apoptosis-inducing Factor (AIF) Isoform with NADH Oxidase Activity

2006 ◽  
Vol 281 (27) ◽  
pp. 18507-18518 ◽  
Author(s):  
Cécile Delettre ◽  
Victor J. Yuste ◽  
Rana S. Moubarak ◽  
Marlène Bras ◽  
Nadine Robert ◽  
...  
Keyword(s):  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Mitochondrial Apoptosis ◽  
Nadh Oxidase Activity ◽  
Apoptosis Inducing Factor ◽  
Identification And Characterization

scholarly journals Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism

2020 ◽  
Author(s):  
Robert J. Nichols ◽  
Benjamin LaFrance ◽  
Naiya R. Phillips ◽  
Luke M. Oltrogge ◽  
Luis E. Valentin-Alvarado ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Biochemical Characterization ◽  
Sulfur Metabolism ◽  
Physiological Role ◽  
Enzymatic Characterization ◽  
Cysteine Desulfurase ◽  
Future Studies ◽  
Pcc 7942

AbstractProkaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryoelectron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria.

Effect of Near-Ultraviolet Light on the Respiratory Chain of Escherichia coli

1971 ◽  
Vol 49 (5) ◽  
pp. 492-495 ◽  
Author(s):  
P. D. Bragg
Keyword(s):  
Ultraviolet Light ◽  
Cytochrome B ◽  
Respiratory Chain ◽  
Nadh Oxidase ◽  
Reductase Activity ◽  
State Level ◽  
Progressive Increase ◽  
E Coli ◽  
Near Ultraviolet ◽  
Nadh Oxidase Activity

Irradiation of a particulate fraction from E. coli with near-ultraviolet light destroyed NADH oxidase activity. This treatment did not affect markedly the levels of cytochromes b1 and o, and ubiquinone in this preparation. Cytochrome a2 was destroyed by irradiation. A progressive increase in the aerobic steady state level of cytochrome b1 reduction during irradiation confirmed that irradiation affected the cytochrome oxidase region of the respiratory chain. There was a second site of inactivation between substrate and cytochrome b1. This was indicated by lowered NADH:cytochrome b1 reductase activity. Partial reactivation of this activity was obtained by addition of ubiqumone-2 but not ubiquinone-8.

scholarly journals Characterization of feline ASCT1 and ASCT2 as RD-114 virus receptor

2013 ◽  
Vol 94 (7) ◽  
pp. 1608-1612 ◽  
Author(s):  
Sayumi Shimode ◽  
Rie Nakaoka ◽  
Hiroko Shogen ◽  
Takayuki Miyazawa
Keyword(s):  
Endogenous Retrovirus ◽  
Amino Acid Transporters ◽  
Extracellular Loop ◽  
Virus Receptor ◽  
Metabolic Role ◽  
Virus Receptors ◽  
Sodium Dependent ◽  
Loop 2

RD-114 virus is a replication-competent feline endogenous retrovirus (ERV). RD-114 virus had been thought to be xenotropic; however, recent findings indicate that RD-114 virus is polytropic and can infect and grow efficiently in feline cells. Receptor(s) for RD-114 virus has not been identified and characterized in cats. In this study, we confirmed that two feline sodium-dependent neutral amino acid transporters (ASCTs), fASCT1 and fASCT2, function as RD-114 virus receptors. By chimeric analyses of feline and murine ASCTs, we revealed that extracellular loop 2 of both fASCT1 and fASCT2 determines the susceptibility to RD-114 virus. Further, we revealed ubiquitous expression of these genes, consistent with the general metabolic role of the ASCT molecules. Our study indicates that RD-114 virus may reinfect tissues and cells in cats, once the virus is activated. Implications of the involvement of RD-114 virus in feline oncogenesis are also discussed.

scholarly journals bis-Molybdopterin Guanine Dinucleotide Is Required for Persistence of Mycobacterium tuberculosis in Guinea Pigs

2014 ◽  
Vol 83 (2) ◽  
pp. 544-550 ◽  
Author(s):  
Monique J. Williams ◽  
Crystal A. Shanley ◽  
Andrew Zilavy ◽  
Blas Peixoto ◽  
Claudia Manca ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrate Reductase ◽  
Guinea Pigs ◽  
Redox Reactions ◽  
Reductase Activity ◽  
Sulfur Metabolism ◽  
Content Type ◽  
Mouse Lungs ◽  
Molybdopterin Guanine Dinucleotide

Mycobacterium tuberculosisis able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo tobis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of themobAhomolog inM. tuberculosiswas confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in amobAdeletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or morebis-MGD-dependent enzymes in the persistence ofM. tuberculosisin guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen.

scholarly journals Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Robert J Nichols ◽  
Benjamin LaFrance ◽  
Naiya R Phillips ◽  
Devon R Radford ◽  
Luke M Oltrogge ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Sulfur Metabolism ◽  
Physiological Role ◽  
Enzymatic Characterization ◽  
Cysteine Desulfurase ◽  
Future Studies ◽  
Cryo Electron Microscopy ◽  
Pcc 7942

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartments in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria.

scholarly journals Characterization of an NADH oxidase of the flavin-dependent disulfide reductase family from Methanocaldococcus jannaschii

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Christopher L. Case ◽  
Jason R. Rodriguez ◽  
Biswarup Mukhopadhyay
Keyword(s):  
Coenzyme A ◽  
Nadh Oxidase ◽  
Specific Activity ◽  
Sulfenic Acid ◽  
Methanocaldococcus Jannaschii ◽  
Nitrobenzoic Acid ◽  
Disulfide Reductase ◽  
Group 3 ◽  
Nadh Oxidase Activity

Methanocaldococcus jannaschii, a deeply rooted hyperthermophilic anaerobic methanarchaeon from a deep-sea hydrothermal vent, carries an NADH oxidase (Nox) homologue (MJ0649). According to the characteristics described here, MJ0649 represents an unusual member within group 3 of the flavin-dependent disulfide reductase (FDR) family. This FDR group comprises Nox, NADH peroxidases (Npx) and coenzyme A disulfide reductases (CoADRs); each carries a Cys residue that forms Cys-sulfenic acid during catalysis. A sequence analysis identified MJ0649 as a CoADR homologue. However, recombinant MJ0649 (rMJNox), expressed in Escherichia coli and purified to homogeneity an 86 kDa homodimer with 0.27 mol FAD (mol subunit)−1, showed Nox but not CoADR activity. Incubation with FAD increased FAD content to 1 mol (mol subunit)−1 and improved NADH oxidase activity 3.4-fold. The FAD-incubated enzyme was characterized further. The optimum pH and temperature were ≥10 and ≥95 °C, respectively. At pH 7 and 83 °C, apparent K m values for NADH and O2 were 3 μM and 1.9 mM, respectively, and the specific activity at 1.4 mM O2 was 60 μmol min−1 mg−1; 62 % of NADH-derived reducing equivalents were recovered as H2O2 and the rest probably generated H2O. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,5′-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high K m, O2 is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.

scholarly journals The NADH oxidase system (external) of muscle mitochondria and its role in the oxidation of cytoplasmic NADH

1985 ◽  
Vol 229 (3) ◽  
pp. 631-641 ◽  
Author(s):  
U F Rasmussen ◽  
H N Rasmussen
Keyword(s):  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Specific Activity ◽  
Reductase Activity ◽  
Heart Mitochondria ◽  
Freezing And Thawing ◽  
Oxidase System ◽  
Muscle Mitochondria ◽  
Nadh Oxidase Activity

An exo-NADH oxidase system [NADH oxidase system (external)], effecting intact-mitochondrial oxidation of added NADH, was studied in pigeon heart mitochondria. Breast muscle mitochondria showed an equal specific activity of the system. The exo-NADH oxidase activity (200 micron mol of NADH/min per g of protein) equalled two-thirds of the State-3 respiratory activity with malate + pyruvate or one-seventh of the total NADH oxidase activity of heart mitochondria. The activity was not caused by use of proteinase in the preparation procedure and all measured parameters were very reproducible from preparation to preparation. The activity is therefore most likely not due to preparation artefacts. The exo-NADH oxidase system is present in all mitochondria in the preparation and is not confined to a subpopulation. The system reduced all cytochrome anaerobically and direct interaction with all cytochrome oxidase was demonstrated by interdependent cyanide inhibition. The exo-NADH oxidase system seems to be located at the outer surface of the mitochondrial inner membrane because, for instance, only this system was rapidly inhibited by rotenone, and ferricyanide could act as acceptor in the rotenone-inhibited system (reductase activity = 20 times oxidase activity). In the presence of antimycin, added NADH reduced only a part of the b-cytochromes. Freezing and thawing the mitochondria, one of the methods used for making them permeable to NADH, destroyed this functional compartmentation. The characteristics of the exo-NADH oxidase system and the malate-aspartate shuttle are compared and the evidence for the shuttle's function in heart in vivo is re-evaluated. It is proposed that oxidation of cytoplasmic NADH in red muscles primarily is effected by the exo-NADH oxidase system.

scholarly journals Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase fromClostridium beijerinckiiNCIMB 8052

2014 ◽  
Vol 80 (6) ◽  
pp. 2011-2020 ◽  
Author(s):  
John Raedts ◽  
Marco A. J. Siemerink ◽  
Mark Levisson ◽  
John van der Oost ◽  
Servé W. M. Kengen
Keyword(s):  
Structural Model ◽  
Reductase Activity ◽  
Physiological Role ◽  
Clostridium Beijerinckii ◽  
Cofactor Specificity ◽  
Threonine Dehydrogenase ◽  
Important Enzyme ◽  
Electronegative Group

ABSTRACTAcetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase fromClostridium beijerinckiiNCIMB 8052. Anin silicoscreen of theC. beijerinckiigenome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression inEscherichia coli. The purified enzyme (C. beijerinckiiacetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the “threonine dehydrogenase and related Zn-dependent dehydrogenases” (COG1063). Metal analysis confirmed the presence of 2 Zn2+atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR inC. beijerinckiiis discussed.

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