vitamin-k-semiquinone-radical and 1-4-dihydroxy-2-naphthoic-acid

Topics: Animals; Bacillus subtilis; Bacteria; Escherichia coli; Genes, Bacterial; Intestines; Ketoglutaric Acids; Lactobacillus; Methylation; Mycobacterium; Naphthols; Oxygen; Phenylbutyrates; Prevotella melaninogenica; Shikimic Acid; Staphylococcus aureus; Vitamin K; Vitamin K 2

A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1, 4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of a menA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from the orf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into the orf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Molecular Sequence Data; Naphthols; Sequence Alignment; Sequence Homology, Amino Acid; Vitamin K

In Escherichia coli, the biosynthesis of the electron carrier menaquinone (vitamin K2) involves at least seven identified enzymatic activities, five of which are encoded in the men cluster. One of these, the conversion of o-succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid, requires the formation of o-succinylbenzoyl-CoA (OSB-CoA) as an intermediate. Formation of the intermediate is mediated by OSB-CoA synthetase encoded by the menE locus known to be located either 5' of menB, or 3' of menC. A DNA fragment overlapping the 3' end of menC in shown by enzymatic complementation to elevate OSB-CoA synthetase activity. Nucleotide sequence analysis of the fragment identified a 1.355-kb open reading frame (ORF) which, when deleted at either the 5' or 3' end, failed to generate increased enzymatic activity. The ORF is preceded by a consensus ribosome-binding site, but no apparent sigma-70 promoter. An oppositely transcribed unidentified gene cluster follows the menE ORF. The region 5' of menB contains an an additional ORF of unknown function (orf241) and establishes the order of genes in the men cluster as menD, orf241, menB, menC and menE. All loci are transcribed counter-clockwise.

Topics: Acyl Coenzyme A; Amino Acid Sequence; Base Sequence; Chromosome Mapping; Cloning, Molecular; Electron Transport; Escherichia coli; Genotype; Molecular Sequence Data; Molecular Structure; Mutation; Naphthols; Open Reading Frames; Operon; Phenylbutyrates; Sequence Analysis, DNA; Sequence Deletion; Succinate-CoA Ligases; Vitamin K

Topics: Bacillus subtilis; Bacteria; Chromates; Coenzyme A; Escherichia coli; Genes, Bacterial; Humans; Mutation; Naphthols; Naphthoquinones; Phenylbutyrates; Pyruvates; Pyruvic Acid; Shikimic Acid; Vitamin K

1,4-Dihydroxy-2-naphthoate:polyprenyltransferase was detected in the membrane fraction from Micrococcus luteus. The specificity of the enzyme ws so tolerant as regards the prenyl-donating substrate that prenyl pyrophosphates ranging in chain length from C15 to C45 were active as substrates. The monophosphate esters were also active, though the reactivities were much lower than those of the corresponding pyrophosphates. The enzyme showed rigorous specificity with respect to the aromatic substrate. Neither 1,4-dihydroxynaphthalene nor its 2-methyl derivative was active at all. 1,4-Dihydroxy-3-methyl-2-naphthoate could be prenylated to afford menaquinone, but the reactivity was much less than that of its demethyl derivative. These results support the view that menaquinone biosynthesis involves the prenylation of 1,4-dihydroxy-2-naphthoate prior to decarboxylation or methylation.

Topics: Alkyl and Aryl Transferases; Cell Membrane; Farnesol; Micrococcus; Naphthols; Polyisoprenyl Phosphates; Sesquiterpenes; Substrate Specificity; Transferases; Vitamin K; Vitamin K 2

Fifteen independent menaquinone biosynthesis mutants (men) of Escherichia coli K12, selected for their inability to use fumarate as terminal electron acceptor, were investigated. Two nutritionally distinct groups were detected. The major group (13 mutants) responded to 1,4-dihydroxy-2-naphthoate (DHN), 2-succinylbenzoate (SB) and its dilactone, whereas the minor group (2 mutants) only responded to DHN. DHN was at least five times more effective than SB but it inhibited growth at concentrations greater than 10 microM. For anaerobic growth on glucose minimal medium the auxotrophs responded to much lower concentrations of DHN and SB and these intermediates could be replaced by uracil. Anaerobic growth tests showed that glycerol, formate and H2 are good substrates for E. coli when fumarate is the ultimate electron acceptor but growth with lactate or with fumarate alone is poor. All 15 men mutations were located between glpT and purF at approximately 49 min in the E. coli linkage map. Cotransduction frequencies with relevant markers were: nalA (21%), glpT (35%) and purF (15%). The presence of at least three genetically distinct classes (menC and menD, SB-requirers; menB, DHN-requirers) was indicated using abortive transduction as a complementation test and three-factor genetic analysis. The relative orientation nalA...menC-(D,B)...purF was indicated. Fluoroacetate-resistant mutants were isolated and four different classes were identified: ack, lacking acetate kinase; pta, lacking phosphotransacetylase; facA, lacking both of these activities; and facB, which retained both of these enzyme activities. Some of the pta mutants and all of the facA mutants failed to grow on media containing fumarate as terminal electron acceptor or anaerobically on glucose minimal medium. All four types had genetic lesions clustered between the men and purF sites. Average cotransduction frequencies with relevant markers were: nalA (4%), men (27 to 35%) and purF (71 to 80%).

Topics: Anaerobiosis; Chromosome Mapping; Escherichia coli; Fluoroacetates; Fumarates; Genes; Genetic Linkage; Mutation; Naphthols; Phenylbutyrates; Vitamin K