Isolation and characterization of a light-sensitive mutant of Escherichia coli K-12 with a mutation in a gene that is required for the biosynthesis of ubiquinone

Nakahigashi, Kenji; Miyamoto, Kazuhito; Nishimura, Koichi; Inokuchi, Hachiro

doi:10.1128/jb.174.22.7352-7359.1992

Cited by 52 publications

(36 citation statements)

References 26 publications

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“…Sequence alignments have classified a number of gene products to flavoprotein hydroxylases (Kahn et al, 1992; Kukor & Olsen, 1992;Nakahigashi et al, 1992;Blanco et al, 1993;Filippini et al, 1995; Haigler et al, 1996;Marin et al, 1996; Seibold et al, 1996;Tsuji et al, 1996;Yang et al, 1996). These sequence data, together with that of PHBH from Pseudomonas jluorescens (van Berkel, 1992), were the starting points for a thorough screening of different databases.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding

Eppink

Schreuder²,

Berkel³

1997

Protein Science

135

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Abstract:A novel conserved sequence motif has been located among the flavoprotein hydroxylases. Based on the crystal structure and site-directed mutagenesis studies of p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas jluorescens, this amino acid fingerprint sequence is proposed to play a dual function in both FAD and NAD(P)H binding. In PHBH, the novel sequence motif (residues 153-166) includes strand A4 and the N-terminal part of helix H7. The conserved amino acids Asp 159, Gly 160, and Arg 166 are necessary for maintaining the structure. The backbone oxygen of Cys 158 and backbone nitrogens of Gly 160 and Phe 161 interact indirectly with the pyrophosphate moiety of FAD, whereas it is known from mutagenesis studies that the side chain of the moderately conserved His 162 is involved in NADPH binding.Keywords: fingerprint; flavoprotein family; NADPH-binding; p-hydroxybenzoate hydroxylase; sequence alignment Flavoprotein hydroxylases are monooxygenases that catalyze the insertion of one atom of molecular oxygen into the substrate, using pyridine nucleotides as external electron donor (van Berkel & Muller, 1991). These enzymes play an important role in the biodegradation of lignin-derived aromatic compounds as well as environmental pollutants, and in the biosynthesis of sterols, antibiotics, and plant hormones. They lack a known fingerprint sequence for NAD(P)H binding, but possess two fingerprint motifs for the FAD binding. The first FAD motif identifies the dinucleotide binding pap-fold, which binds the ADP moiety of FAD (Wierenga et al., 1986), whereas the second motif represents residues that are in contact with the riboflavin moiety of FAD (Eggink et al., 1990).PHBH (EC 1.14.13.2) is the prototype of FAD-dependent hydroxylases, and the only enzyme in this class of flavoproteins for which a three-dimensional structure is known in atomic detail .In the past few years, the number of flavoprotein hydroxylase cloned genes has increased tremendously, and about 50 amino acid sequences are known currently. Therefore, and in view of the unknown binding mode of NADPH in this class of flavoenzymes, it was of interest to search for the presence of conserved sequence motifs. This report describes the identification of a novel sequence motif in flavoprotein hydroxylases, which appears to be important for the binding of both FAD and NAD(P)H. Discussion:Sequence alignments have classified a number of gene products to flavoprotein hydroxylases (Kahn et al., 1992; Kukor & Olsen, 1992;Nakahigashi et al., 1992;Blanco et al., 1993;Filippini et al., 1995; Haigler et al., 1996;Marin et al., 1996; Seibold et al., 1996;Tsuji et al., 1996;Yang et al., 1996). These sequence data, together with that of PHBH from Pseudomonas jluorescens (van Berkel, 1992), were the starting points for a thorough screening of different databases. This search was performed with BEAUTY, which is an BLAST-enhanced alignment utility that integrates multiple biological information resources (Worley et al., 1995). From the 50 collected sequences, small ...

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Section: Discussionmentioning

confidence: 99%

Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding

Eppink

Schreuder²,

Berkel³

1997

Protein Science

135

View full text Add to dashboard Cite

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“…42). The ubiH gene product was identified as a flavin-dependent monooxygenase (43). The E. coli ubiF gene product has also been identified as a flavin-dependent monooxygenase, with 31% amino acid sequence identity to UbiH (44).…”

Section: Coq6 Is a Flavin-dependent Monooxygenase In Q Biosynthesismentioning

confidence: 99%

The Saccharomyces cerevisiae COQ6 Gene Encodes a Mitochondrial Flavin-dependent Monooxygenase Required for Coenzyme Q Biosynthesis

Gin

Hsu

Rothman

et al. 2003

Journal of Biological Chemistry

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Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8. Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene. coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid. The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner. Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane. Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavindependent monooxygenase required for one or more steps in Q biosynthesis.

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“…Genes with unknown function that were found to be associated with persistence are visC, yacC, and yiiS (Table S1). VisC is a predicted oxidoreductase with an FAD/NAD(P)-binding domain; however, deletion of visC has been found to have no effect on aerobic respiration (25). The gene products of yacC and yiiS have no assigned function, although, yiiS has been identified as a member of the sigma E regulon (26).…”

Section: Increased Persistence Through the Accumulation Of The Intracmentioning

confidence: 99%

Large mutational target size for rapid emergence of bacterial persistence

Girgis

Harris²,

Tavazoie³

2012

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

114

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Phenotypic heterogeneity displayed by a clonal bacterial population permits a small fraction of cells to survive prolonged exposure to antibiotics. Although first described over 60 y ago, the molecular mechanisms underlying this behavior, termed persistence, remain largely unknown. To systematically explore the genetic basis of persistence, we selected a library of transposon-mutagenized Escherichia coli cells for survival to multiple rounds of lethal ampicillin exposure. Application of microarray-based genetic footprinting revealed a large number of loci that drastically elevate persistence frequency through null mutations and domain disruptions. In one case, the C-terminal disruption of methionyl-tRNA synthetase (MetG) results in a 10,000-fold higher persistence frequency than wild type. We discovered a mechanism by which null mutations in transketolase A ( tktA ) and glycerol-3-phosphate (G3P) dehydrogenase ( glpD ) increase persistence through metabolic flux alterations that increase intracellular levels of the growth-inhibitory metabolite methylglyoxal. Systematic double-mutant analyses revealed the genetic network context in which such persistent mutants function. Our findings reveal a large mutational target size for increasing persistence frequency, which has fundamental implications for the emergence of antibiotic tolerance in the clinical setting.

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Isolation and characterization of a light-sensitive mutant of Escherichia coli K-12 with a mutation in a gene that is required for the biosynthesis of ubiquinone

Cited by 52 publications

References 26 publications

Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding

Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding

The Saccharomyces cerevisiae COQ6 Gene Encodes a Mitochondrial Flavin-dependent Monooxygenase Required for Coenzyme Q Biosynthesis

Large mutational target size for rapid emergence of bacterial persistence

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