Dataset Information

Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menA gene from Escherichia coli.

ABSTRACT: 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.

SUBMITTER: Suvarna K

PROVIDER: S-EPMC107237 | biostudies-literature | 1998 May

REPOSITORIES: biostudies-literature

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Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menA gene from Escherichia coli.

Suvarna K K Stevenson D D Meganathan R R Hudspeth M E ME

Journal of bacteriology 19980501 10

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 ...[more]

PMID: 9573170

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Project description:Strains of Escherichia coli with mutations in the ubiE gene are not able to catalyze the carbon methylation reaction in the biosynthesis of ubiquinone (coenzyme Q) and menaquinone (vitamin K2), essential isoprenoid quinone components of the respiratory electron transport chain. This gene has been mapped to 86 min on the chromosome, a region where the nucleic acid sequence has recently been determined. To identify the ubiE gene, we evaluated the amino acid sequences encoded by open reading frames located in this region for the presence of sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases. One open reading frame in this region (o251) was found to encode these motifs, and several lines of evidence that confirm the identity of the o251 product as UbiE are presented. The transformation of a strain harboring the ubiE401 mutation with o251 on an expression plasmid restored both the growth of this strain on succinate and its ability to synthesize both ubiquinone and menaquinone. Disruption of o251 in a wild-type parental strain produced a mutant with defects in growth on succinate and in both ubiquinone and menaquinone synthesis. DNA sequence analysis of the ubiE401 allele identified a missense mutation resulting in the amino acid substitution of Asp for Gly142. E. coli strains containing either the disruption or the point mutation in ubiE accumulated 2-octaprenyl-6-methoxy-1,4-benzoquinone and demethylmenaquinone as predominant intermediates. A search of the gene databases identified ubiE homologs in Saccharomyces cerevisiae, Caenorhabditis elegans, Leishmania donovani, Lactococcus lactis, and Bacillus subtilis. In B. subtilis the ubiE homolog is likely to be required for menaquinone biosynthesis and is located within the gerC gene cluster, known to be involved in spore germination and normal vegetative growth. The data presented identify the E. coli UbiE polypeptide and provide evidence that it is required for the C methylation reactions in both ubiquinone and menaquinone biosynthesis.

Project description:Human hepatocellular carcinoma cells, HepG2 , cultured in DMEM containing 10% fetal calf serum. To study gene expression changes induced by Vitamin K2, total RNA was isolated following the Isogen procedure (Nippon-gene, Tokyo, Japan) from HepG2 cells with and without 50 microM of Vitamin K2 treatment for three days. The cDNA microarrays Human Oligo Chip 30K “AceGene” subset A was purchased from Hitachi Software Engineering Co. (Yokohama-City, Japan). This array contains 10368 kinds of gene-specific 50 mer sense oligonucleotides. Subset A contains mainly known function ORF oligo probes. Preparation of fluorescent cDNA targets by an indirect labeling approach was performed using PowerScript reverse transcription kit (Clontech). First-strand cDNAs were synthesized from 30 microg of total RNA from HepG2 cells treated with or without Vitamin K2 using random primer and PoweScript Fluorescent Labelling Kit. Monofunctional, N-hydroxysuccinimide-activated fluorescent dyes (Cy3 and Cy5; Amersham) were coupled to the cDNAs by reaction with the amino functional groups. Untreated cells were labeled with Cy3 as a control and treated cells with Cy5 as a sample. After free dyes were removed using MinElute Purification kit (Qiagen), the targets were mixed together and added to the microarrays, and then incubated overnight (16 hours) at 42?C. The slides were washed at 30?C in each with 2x SSC, 0.1% SDS and 2x SSC for 10 min and 5 min, respectively, and washed in 1x SSC for 5 min. Fluorescent images of the hybridized microarrays were scanned with a fluorescence laser confocal slide scanner (Affymetrix 428 Array Scanner, Affymetrix, Santa Clara, CA). The Cy3 and Cy5 intensities with background subtraction were determined by ImaGene 4.2 software (BioDiscovery, Marina Del Rey, CA). The expression data were then filtered based on their channel intensities, spot size and flag (missing or inaccurate data), and the Cy5/Cy3 ratios were calculated and normalized by median-centering the log-ratio of all genes.

Dataset Information

Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menA gene from Escherichia coli.

Publications

Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menA gene from Escherichia coli.

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