The mechanism of catalysis by type-II NADH:quinone oxidoreductases

Blaza, James N.; Bridges, Hannah R.; Aragão, D.; Dunn, Elyse; Heikal, Adam; Cook, Gregory M.; Nakatani, Yoshio; Hirst, Judy

doi:10.1038/srep40165

Cited by 56 publications

(93 citation statements)

References 41 publications

(74 reference statements)

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“…Currently, some of the described FAD/NADH-dependent oxidoreductases are considered in microbiology a crucial target of antibiotics or chemotherapeutics (i.e., those structurally related to RYL-552, a quinolinic derivative or to SL827, a sulphonamide derivative [10,48,[51][52][53]) because those enzymes play a fundamental role for the ATP synthesis and oxidative pathways in most microorganisms. The lack of NDH-2-orthologous enzymes in Mammalia was retained an important proof of the selective action of potential drug development [10,[54][55][56][57].…”

Section: 2mentioning

confidence: 99%

FAD/NADH Dependent Oxidoreductases: From Different Amino Acid Sequences to Similar Protein Shapes for Playing an Ancient Function

Trisolini

Gambacorta

Gorgoglione

et al. 2019

JCM

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Flavoprotein oxidoreductases are members of a large protein family of specialized dehydrogenases, which include type II NADH dehydrogenase, pyridine nucleotide-disulphide oxidoreductases, ferredoxin-NAD+ reductases, NADH oxidases, and NADH peroxidases, playing a crucial role in the metabolism of several prokaryotes and eukaryotes. Although several studies have been performed on single members or protein subgroups of flavoprotein oxidoreductases, a comprehensive analysis on structure–function relationships among the different members and subgroups of this great dehydrogenase family is still missing. Here, we present a structural comparative analysis showing that the investigated flavoprotein oxidoreductases have a highly similar overall structure, although the investigated dehydrogenases are quite different in functional annotations and global amino acid composition. The different functional annotation is ascribed to their participation in species-specific metabolic pathways based on the same biochemical reaction, i.e., the oxidation of specific cofactors, like NADH and FADH2. Notably, the performed comparative analysis sheds light on conserved sequence features that reflect very similar oxidation mechanisms, conserved among flavoprotein oxidoreductases belonging to phylogenetically distant species, as the bacterial type II NADH dehydrogenases and the mammalian apoptosis-inducing factor protein, until now retained as unique protein entities in Bacteria/Fungi or Animals, respectively. Furthermore, the presented computational analyses will allow consideration of FAD/NADH oxidoreductases as a possible target of new small molecules to be used as modulators of mitochondrial respiration for patients affected by rare diseases or cancer showing mitochondrial dysfunction, or antibiotics for treating bacterial/fungal/protista infections.

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Section: 2mentioning

confidence: 99%

FAD/NADH Dependent Oxidoreductases: From Different Amino Acid Sequences to Similar Protein Shapes for Playing an Ancient Function

Trisolini

Gambacorta

Gorgoglione

et al. 2019

JCM

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“…Low iron also resulted in a 1.7 LFC of PA2691 ( Supplementary Dataset S1 ), which is a predicted type II NADH:quinone oxidoreductase (NDH-2). NDH-2 proteins utilize flavin cofactors instead of iron to catalyze the oxidation of NADH and reduce quinones (39). Thus, PA2691 may function in place of the nuoA-N encoded complex I to oxidize NADH under low iron conditions.…”

Section: Resultsmentioning

confidence: 99%

“…It was previously shown that cyoA expression increases under low iron conditions via loss of Fur repression (31), most likely due to requiring less iron than the other four oxidases. Under iron-replete conditions, the NADH dehydrogenase I, comprised of NuoA-N, is the predominant NADH dehydrogenase as it not only recycles NADH to NAD + but also helps to generate a proton motive force that can be used to generate ATP (39). Likewise, the cbb 3 -type cytochrome oxidases are the predominant terminal oxidases, as they interact with the cytochrome bc 1 complex, which also contributes to the proton motive force (63).…”

Section: Discussionmentioning

confidence: 99%

Proteomic analysis of thePseudomonas aeruginosairon starvation response reveals PrrF sRNA-dependent regulation of amino acid metabolism, iron-sulfur cluster biogenesis, motility, and zinc homeostasis

Nelson

Huang

et al. 2018

Preprint

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Iron is a critical nutrient for most microbial pathogens, and the innate immune system exploits this requirement by sequestering iron and other metals through a process termed nutritional immunity. The opportunistic pathogen Pseudomonas aeruginosa provides a model system for understanding the microbial response to host iron depletion, as this organism exhibits a high requirement for iron as well as an exquisite ability to overcome iron deprivation during infection. Hallmarks of P. aeruginosa’s iron starvation response include the induction of multiple high affinity iron acquisition systems and an “iron sparing response” that is post-transcriptionally mediated by the PrrF small regulatory RNAs (sRNAs). Here, we used liquid chromatography-tandem mass spectrometry to conduct label-free proteomics of the P. aeruginosa iron starvation response, revealing several iron-regulated processes that have not been previously described. Iron starvation induced multiple proteins involved in branched chain and aromatic amino acid catabolism, providing the capacity for iron-independent entry of carbons into the TCA cycle. Proteins involved in sulfur assimilation and cysteine biosynthesis were reduced upon iron starvation, while proteins involved in iron-sulfur cluster biogenesis were paradoxically increased, highlighting the central role of iron in P. aeruginosa metabolism. Iron starvation also resulted in changes in the expression of several zinc-responsive proteins, as well as the first experimental evidence for increased levels of twitching motility proteins upon iron starvation. Subsequent proteomics analyses demonstrated that the PrrF sRNAs were required for iron regulation of many of these newly-identified proteins, and we identified PrrF complementarity with mRNAs encoding key iron-regulated proteins involved in amino acid metabolism, iron-sulfur biogenesis, and zinc homeostasis. Combined, these results provide the most comprehensive view of the P. aeruginosa iron starvation response to date and outline novel roles for the PrrF sRNAs in the P. aeruginosa iron sparing response and pathogenesis.AUTHOR SUMMARYIron is central for the metabolism of almost all microbial pathogens, and as such this element is sequestered by the host innate immune system to restrict microbial growth. Defining the response of microbial pathogens to iron starvation is therefore critical for understanding how pathogens colonize and propagate within the host. The opportunistic pathogen Pseudomonas aeruginosa, which causes significant morbidity and mortality in compromised individuals, provides an excellent model for studying this response due to its high requirement for iron yet well-documented ability to overcome iron starvation. Here we used label-free proteomics to investigate the P. aeruginosa iron starvation response, revealing a broad landscape of metabolic and metal homeostasis changes that have not previously been described. We further provide evidence that many of these processes are regulated through the iron responsive PrrF small regulatory RNAs, which are integral to P. aeruginosa iron homeostasis and virulence. These results demonstrate the power of proteomics for defining stress responses of microbial pathogens, and they provide the most comprehensive analysis to date of the P. aeruginosa iron starvation response.

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“…S8) showed a decrease in flavin absorbance at 456 nm and an increase in absorption in the 500 -700 nm region. The decrease of flavin absorption is caused by electron transfer from NADH to the oxidized flavin moiety, whereas formation of the species that absorbs at long wavelengths is caused by formation of a charge-transfer complex (HadX FADHϪ -NAD ϩ ) similar to what has been observed dur-ing the reductive half-reactions of many flavin reductases (21,31,35,43). When similar experiments using NADPH were carried out, we could not detect any reduction of the enzyme-bound flavin by NADPH within 30 s (data not shown), indicating that HadX is only specific for using NADH as a reductant.…”

Section: Kinetics Of Flavin Reduction On Hadxmentioning

confidence: 94%

A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin–dependent enzymes

Pimviriyakul

Chaiyen

2018

Journal of Biological Chemistry

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Edited by F. Peter GuengerichHalogenated phenol and nitrophenols are toxic compounds that are widely accumulated in the environment. Enzymes in the had operon from the bacterium Ralstonia pickettii DTP0602 have the potential for application as biocatalysts in the degradation of many of these toxic chemicals. HadA monooxygenase previously was identified as a two-component reduced FAD (FADH ؊ )-utilizing monooxygenase with dual activities of dehalogenation and denitration. However, the partner enzymes of HadA, that is, the flavin reductase and quinone reductase that provide the FADH ؊ for HadA and reduce quinone to hydroquinone, remain to be identified. In this report, we overexpressed and purified the flavin reductases, HadB and HadX, to investigate their functional and catalytic properties. Our results indicated that HadB is an FMN-dependent quinone reductase that converts the quinone products from HadA to hydroquinone compounds that are more stable and can be assimilated by downstream enzymes in the pathway. Transient kinetics indicated that HadB prefers NADH and menadione as the electron donor and acceptor, respectively. We found that HadX is an FAD-bound flavin reductase, which can generate FADH ؊ for HadA to catalyze dehalogenation or denitration reactions. Thermodynamic and transient kinetic experiments revealed that HadX prefers to bind FAD over FADH؊ and that HadX can transfer FADH ؊ from HadX to HadA via free diffusion. Moreover, HadX rapidly catalyzed NADH-mediated reduction of flavin and provided the FADH ؊ for a monooxygenase of a different system. Combination of all three flavin-dependent enzymes, i.e. HadA/HadB/HadX, reconstituted an effective dehalogenation and denitration cascade, which may be useful for future bioremediation applications. 2 The abbreviations used are: HPs, halogenated phenol; HadA, two-component FADH Ϫ utilizing monooxygenase; HadB, a quinone reductase encoded in the had operon; HadX, a flavin reductase encoded in the had operon; NPs, nitrophenols; FADH Ϫ , reduced flavin adenine dinucleotide; FMNH Ϫ , reduced flavin mononucleotide; 4-NP, 4-nitrophenol; 4-CP, 4-chlorophenol; HQ, hydroquinone; BQ, benzoquinone; C 1 , the flavin reductase component of p-hydroxyphenylacetate 3-hydroxylase from A. buamannii; C 2 , the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase from A.

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The mechanism of catalysis by type-II NADH:quinone oxidoreductases

Cited by 56 publications

References 41 publications

FAD/NADH Dependent Oxidoreductases: From Different Amino Acid Sequences to Similar Protein Shapes for Playing an Ancient Function

FAD/NADH Dependent Oxidoreductases: From Different Amino Acid Sequences to Similar Protein Shapes for Playing an Ancient Function

Proteomic analysis of thePseudomonas aeruginosairon starvation response reveals PrrF sRNA-dependent regulation of amino acid metabolism, iron-sulfur cluster biogenesis, motility, and zinc homeostasis

A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin–dependent enzymes

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