Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis

Kwon, Ohsuk; Hudspeth, Michael E. S.; Meganathan, R.

doi:10.1128/jb.178.11.3252-3259.1996

Cited by 41 publications

(29 citation statements)

References 33 publications

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“…Consistent with this observation is the recent demonstration that enterobactin is synthesized both aerobically and anaerobically (6). Since menaquinone is synthesized both aerobically and anaerobically (at a higher level) and functions anaerobically, the reaction carried out by MenF will be similar in the utilization of water as the source of the hydroxyl group.…”

supporting

confidence: 69%

“…The various enzymes involved in the menaquinone biosynthetic pathway are present at extremely low levels, and among these, the menF-encoded isochorismate synthase is the least active. In the entC mutant (which lacks the EntC isochorismate synthase), the MenF activity was reported to be 0.6 nmol h Ϫ1 mg of protein Ϫ1 (6). The strain in which the protein was overexpressed produced 45 nmol of isochorismate min Ϫ1 mg of protein…”

Section: Discussionmentioning

confidence: 99%

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Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli

et al. 1997

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The first committed step in the biosynthesis of menaquinone (vitamin K 2 ) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene. This isochorismate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in enterobactin biosynthesis. MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity. The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 48 kDa. Thus, the enzyme is a homodimer. The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37°C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg . In the presence of 30 mM ␤-mercaptoethanol (BME), the k cat increased to 176 min ؊1 . The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold. Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity. Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted in complete protection of the enzyme from inactivation.

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supporting

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli

et al. 1997

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“…The mutant was able to grow at the same rate as the parent, even in the presence of high concentrations of iron. Further, the mutant produced as much MK as the parent (91,93,104). These results provided clear evidence for the presence of an alternate isochorismate synthase specifically involved in MK biosynthesis.…”

Section: Formation Of Isochorismate (Compound IV -------> V)supporting

confidence: 53%

“…To study the regulation of the entC gene, an entC-lacZ operon fusion was constructed and the expression of β-galactosidase monitored under various conditions. It was found that the β-galactosidase was fully derepressed at low concentration of iron and repressed at high iron concentrations under both aerobic and anaerobic growth conditions (91,93).…”

Section: Formation Of Isochorismate (Compound IV -------> V)mentioning

confidence: 99%

Biosynthesis of Menaquinone (Vitamin K2) and Ubiquinone (Coenzyme Q)

Meganathan¹,

Kwon²

2009

EcoSal

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“…To construct ⌽(arcB -malF [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] arcB ), PCR was performed with pIBW as a template and BPH-N and BMF-22 as primers. The purified PCR product and BMF-35 were used as primers for the second PCR with pDHB32 (6) as a template.…”

Section: Fig 3 Effects Of Substituting Segments Of Arcb Withmentioning

confidence: 99%

The ArcB Sensor Kinase of Escherichia coli : Genetic Exploration of the Transmembrane Region

Kwon

Georgellis

Lynch³

et al. 2000

J Bacteriol

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The Arc two-component signal transduction system of Escherichia coli regulates the expression of numerous operons in response to respiratory growth conditions. Cellular redox state or proton motive force (⌬ H ؉) has been proposed to be the signal for the membrane-associated ArcB sensor kinase. This study provided evidence for a short ArcB periplasmic bridge that contains a His47. The dispensability of this amino acid, the only amino acid with a pK in the physiological range, renders the ⌬ H ؉ model unlikely. Furthermore, results from substituting membrane segments of ArcB with counterparts of MalF indicate that the region does not play a stereospecific role in signal reception.The Arc two-component signal transduction system of Escherichia coli regulates the expression of more than 30 operons, depending on the redox conditions of growth (20,22,30,31). The system consists of ArcB, the membrane-bound sensor kinase, and ArcA, the cognate response regulator. ArcB (17,23,27,48) (see Fig. 1A) belongs to the tripartite sensor kinase subfamily (1,16,25,35,37,44,49), is attached to the cytoplasmic membrane by two transmembrane segments (TM1 and TM2) near the N-terminal end (24), and catalyzes a phosphorelay via His292, Asp576, and His717 of ArcB to Asp54 of ArcA (14). The autophosphorylation step is stimulated by effectors, such as D-lactate, pyruvate, and acetate. These metabolites accumulate when exogenous electron acceptors limit respiration during growth (13, 18). Dephosphorylation of ArcA-P occurs by a reverse phosphorelay from the Asp54 of ArcA to the His717 and Asp576 of ArcB. The phosphoryl group is then released as P i (12).Most sensor kinases receive their signal from the periplasmic domain, resulting in conformation changes that trigger autophosphorylation. As a sensor kinase, ArcB is unusual in having a short putative periplasmic bridge (24,29). Relatively little is known about the nature of the signal for ArcB and what role the membrane-associated region plays in signal reception, except that autophosphorylation seems to be activated by excessive reducing equivalents (7,11,21,22,24,38). Two studies involving growth of cells at high pH and treatment of cells by protonophores during growth, however, led to the suggestion that ArcB kinase is activated by a decrease in proton motive force (PMF) across the cytoplasmic membrane (2, 5). Here, we confirm the transmembrane topology of ArcB by genetic analysis and probe the function of the membrane region by replacing the chromosomal arcB ϩ by a single copy of a mutant allele ( Fig. 2; Tables 1 and 2).Transmembrane topology of ArcB. To test the suggested topology based on hydrophobicity analysis, we constructed four phoA protein fusions (32) of arcB (Fig. 1B). The PhoA fusions at residues 22 and 102 of ArcB exhibited very low levels of alkaline phosphatase activity. In contrast, the PhoA fusions at residues 41 and 57 of ArcB showed very high levels of the enzyme activity (Fig. 1B). On the basis of this genetic analysis and a more recent algorithm for determining membr...

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Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis

Cited by 41 publications

References 33 publications

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli

Biosynthesis of Menaquinone (Vitamin K2) and Ubiquinone (Coenzyme Q)

The ArcB Sensor Kinase of Escherichia coli : Genetic Exploration of the Transmembrane Region

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