Aeration‐dependent changes in composition of the quinone pool in <i>Escherichia coli</i>

To understand the biochemical basis for the function of the rotenone-insensitive internal NADH-quinone (Q) oxidoreductase (Ndi1), we have overexpressed mature Ndi1 in Escherichia coli membranes. The Ndi1 purified from the membranes contained one FAD and showed enzymatic activities comparable with the original Ndi1 isolated from Saccharomyces cerevisiae. When extracted with Triton X-100, the isolated Ndi1 did not contain Q. The Q-bound form was easily reconstituted by incubation of the Q-free Ndi1 enzyme with ubiquinone-6. We compared the properties of Q-bound Ndi1 enzyme with those of Q-free Ndi1 enzyme, with higher activity found in the Q-bound enzyme. Although both are inhibited by low concentrations of AC0 -11 (IC 50 ‫؍‬ 0.2 M), the inhibitory mode of AC0 -11 on Q-bound Ndi1 was distinct from that of Q-free Ndi1. The bound Q was slowly released from Ndi1 by treatment with NADH or dithionite under anaerobic conditions. This release of Q was prevented when Ndi1 was kept in the reduced state by NADH. When Ndi1 was incorporated into bovine heart submitochondrial particles, the Q-bound form, but not the Q-free form, established the NADH-linked respiratory activity, which was insensitive to piericidin A but inhibited by KCN. Furthermore, Ndi1 produces H 2 O 2 as isolated regardless of the presence of bound Q, and this H 2 O 2 was eliminated when the Q-bound Ndi1, but not the Q-free Ndi1, was incorporated into submitochondrial particles. The data suggest that Ndi1 bears at least two distinct Q sites: one for bound Q and the other for catalytic Q. Alternative NADH dehydrogenases (NDH-2)3 catalyze electron transfer from NADH to quinone (Q) without the energy transduction. NDH-2 is considered to be composed of a single polypeptide, houses FAD as a cofactor (1, 2) and is regarded to have the simplest structure among the NADH dehydrogenases (2). The NDH-2 enzymes are present in bacteria, plant, and fungal mitochondria but not in mammalian mitochondria. In bacteria, all NDH-2 enzymes known to date are located in the cytoplasmic phase. By contrast, plant and fungal mitochondria possess two types of NDH-2 enzymes (3); one is directed to the matrix and catalyzes NADH oxidation in the matrix (designated the internal NADH dehydrogenase or Ndi), whereas the other faces the intermembrane space and oxidizes NADH in the cytoplasmic space (designated the external NADH dehydrogenases or Nde). The Ndi1 enzyme is similar to complex I in terms of NADH oxidation in the matrix (4).A series of studies in our laboratory suggest that the Saccharomyces cerevisiae NDI1 gene may work as a therapeutic agent for mitochondrial diseases caused by complex I deficiencies (5-13). In fact, the Ndi1 expression in the substantia nigra of mouse brains has protective effects against Parkinsonian symptoms caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment (12,14). Recently, we illustrated that the expressed Ndi1 enzyme may play a dual role in rescuing complex I-deficient cells (15); one is to restore the NADH oxidase activity, and the other i...

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“…It is known that UQ 8 and MQ 8 are major Q components in E. coli (37). We examined whether the purified Ndi1 contains Q and which Q if it does.…”

Section: Resultsmentioning

confidence: 99%

Roles of Bound Quinone in the Single Subunit NADH-Quinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae

Yamashita

Nakamaru‐Ogiso

Miyoshi

et al. 2007

Journal of Biological Chemistry

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“…It is not clear why these synthases are evolved into two evolutionary groups with distinct bicarbonate dependence. However, it is noticeable that the bicarbonateactivatable EcMenB is the only enzyme subjected to external control in the menaquinone biosynthetic pathway of E. coli, which may be linked to the proposed regulation of the pathway by post-transcriptional modulation of enzymatic activity (19). Due to this potential connection, it may be worthwhile to investigate the correlation of menaquinone/phylloquinone biosynthesis to intracellular bicarbonate level in other species utilizing the type I DHNA-CoA synthases.…”

Section: Discussionmentioning

confidence: 99%

“…The naphthoquinone level is significantly up-regulated during the aerobiosis-to-anaerobiosis transition (15-17), indicating that menaquinone biosynthesis is subject to aeration control. However, the O 2 -dependent Arc, Fnr, and AppY transcriptional regulatory systems have been demonstrated not to regulate the biosynthesis of menaquinone by gene knock-out experiments (18,19). In addition, global transcription suppression by chloramphenicol has no effect on the anaerobiosis-induced menaquinone biosynthesis, suggesting that the biosynthesis is controlled post-transcriptionally via enzymatic activity modulation (19).…”

mentioning

confidence: 99%

“…However, the O 2 -dependent Arc, Fnr, and AppY transcriptional regulatory systems have been demonstrated not to regulate the biosynthesis of menaquinone by gene knock-out experiments (18,19). In addition, global transcription suppression by chloramphenicol has no effect on the anaerobiosis-induced menaquinone biosynthesis, suggesting that the biosynthesis is controlled post-transcriptionally via enzymatic activity modulation (19). So far, no enzyme has been identified to be involved in such a regulation system.…”

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confidence: 99%

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A Bicarbonate Cofactor Modulates 1,4-Dihydroxy-2-naphthoyl-Coenzyme A Synthase in Menaquinone Biosynthesis of Escherichia coli

Jiang

Chen

Guo

et al. 2010

Journal of Biological Chemistry

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1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzymebound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the ␣-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases.Menaquinone is a lipid soluble naphthoquinone that plays important biological roles. In many bacterial microorganisms, menaquinone serves as an electron transporter in the respiratory chain and is essential for their survival (1, 2). In humans and animals, menaquinone is a vitamin (K2) and is used as an enzyme cofactor involved in post-translational glutamate ␥-carboxylation of proteins in blood coagulation (3, 4), bone metabolism (5), and calcification of arteries and other soft tissues (6). This naphthoquinone is synthesized from the chorismate of the shikimate pathway in microorganisms (1, 2, 7, 8), whereas it has to be acquired from diet or intestinal microflora in humans or animals (9, 10). Due to its absence in humans and animals, menaquinone biosynthesis has been an attractive target for development of antibiotics against a number of important microbial pathogens, such as Mycobacterium tuberculosis and Staphylococcus aureus (11,12).Menaquinone biosynthesis has been most extensively studied in the facultative anaerobe Escherichia coli that uses the naphthoquinone (MK-8) and its demethylated congener (demethylmenaquinone, DMK-8) as an obligatory electron transporter under anaerobic conditions when the electron acceptor is fumarate, dimethyl sulfoxide, or trimethylamine N-oxide (13,14). The naphthoquinone level is significantly up-regulated during the aerobiosis-to-anaerobiosis transition (15-17), indicating that menaquinone biosynthesis is subject to aerati...

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“…Detection of the quinones was carried out at 290 nm for ubiquinones, at 248 nm for menaquinones, and at 270 nm to record all quinones simultaneously. The amounts of all quinones were calculated from the peak areas using UQ 10 and MK 4 as standards according to the method applied by Shestopalov et al (51). Methanol, ethanol, and petroleum ether were of analytical grade.…”

Section: Methodsmentioning

confidence: 99%

ErpA, an iron–sulfur (Fe–S) protein of the A-type essential for respiratory metabolism in Escherichia coli

Loiseau

Gerez

Bekker

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

135

149

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Understanding the biogenesis of iron-sulfur (Fe-S) proteins is relevant to many fields, including bioenergetics, gene regulation, and cancer research. Several multiprotein complexes assisting Fe-S assembly have been identified in both prokaryotes and eukaryotes. Here, we identify in Escherichia coli an A-type Fe-S protein that we named ErpA. Remarkably, erpA was found essential for growth of E. coli in the presence of oxygen or alternative electron acceptors. It was concluded that isoprenoid biosynthesis was impaired by the erpA mutation. First, the eukaryotic mevalonate-dependent pathway for biosynthesis of isopentenyl diphosphate restored the respiratory defects of an erpA mutant. Second, the erpA mutant contained a greatly reduced amount of ubiquinone and menaquinone. Third, ErpA bound Fe-S clusters and transferred them to apo-IspG, a protein catalyzing isopentenyl diphosphate biosynthesis in E. coli. Surprisingly, the erpA gene maps at a distance from any other Fe-S biogenesis-related gene. ErpA is an A-type Fe-S protein that is characterized by an essential role in cellular metabolism.essential gene yadR ͉ isoprenoid ͉ respiration ͉ scaffold

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Aeration‐dependent changes in composition of the quinone pool in Escherichia coli

Cited by 66 publications

References 18 publications

Roles of Bound Quinone in the Single Subunit NADH-Quinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae

Roles of Bound Quinone in the Single Subunit NADH-Quinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae

A Bicarbonate Cofactor Modulates 1,4-Dihydroxy-2-naphthoyl-Coenzyme A Synthase in Menaquinone Biosynthesis of Escherichia coli

ErpA, an iron–sulfur (Fe–S) protein of the A-type essential for respiratory metabolism in Escherichia coli

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