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

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 is 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 σ⁷⁰ promoter. An oppositely transcribed unidentified gene cluster follows the menE ORF. The region 5′ of menB contains 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.     

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from Escherichia coli.

The coenzyme A (CoA)- and ATP-dependent conversion of o-succinylbenzoic acid [OSB; 4-(2'-carboxyphenyl)-4-oxobutyric acid], to o-succinylbenzoyl-CoA is carried out by the enzyme o-succinylbenzoyl-CoA synthetase. o-Succinylbenzoyl-CoA is a key intermediate in the biosynthesis of menaquinone (vitamin K2) in both gram-negative and gram-positive bacteria. The enzyme has been overexpressed and purified to homogeneity. The purified enzyme was found to have a native molecular mass of 185 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular mass of 49 kDa. Thus, the enzyme is a homotetramer. The enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 30 to 40 degrees C. The Km values for OSB, ATP, and CoA were 16, 73.5, and 360 microM, respectively. Of the various metal ions tested, Mg2+ was found to be the most effective in stimulating the enzyme activity. Studies with substrate analogs showed that neither benzoic acid nor benzoylpropionic acid (succinylbenzene) is a substrate for the enzyme. Thus, it appears that both the benzoyl carboxyl group and the succinyl side chain are required for activation of the aliphatic carboxyl group.     

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

The first committed step in the biosynthesis of menaquinone (vitamin K2) 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 degrees C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg2+. The enzyme was found to have a Km of 195 +/- 23 microM and a k(cat) of 80 min(-1). In the presence of 30 mM beta-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.     

Menaquinone (vitamin K2) biosynthesis: evidence that the Escherichia coli menD gene encodes both 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase and alpha-ketoglutarate decarboxylase activities.

The formation of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC), the first identified intermediate in the menaquinone biosynthetic pathway, requires two reactions. They are the decarboxylation of alpha-ketoglutarate by an alpha-ketoglutarate decarboxylase, which results in the formation of succinic semialdehyde-thiamine PPi (TPP) anion, and the addition of the succinic semialdehyde-TPP anion to isochorismate carried out by the enzyme SHCHC synthase. Evidence is provided to support the conclusion that both enzymatic activities are encoded by an extended menD gene which is capable of generating a bifunctional 69-kDa protein. Consistent with the requirement for TPP in the decarboxylation of alpha-ketoglutarate, the translated amino acid sequence contains the characteristic TPP-binding motif present in all well-characterized TPP-requiring enzymes.     

Evolution of enzymatic activity in the enolase superfamily: structure of o-succinylbenzoate synthase from Escherichia coli in complex with Mg2+ and o-succinylbenzoate

The X-ray structures of the ligand free (apo) and the Mg(2+)*o-succinylbenzoate (OSB) product complex of o-succinylbenzoate synthase (OSBS) from Escherichia coli have been solved to 1.65 and 1.77 A resolution, respectively. The structure of apo OSBS was solved by multiple isomorphous replacement in space group P2(1)2(1)2(1); the structure of the complex with Mg(2+)*OSB was solved by molecular replacement in space group P2(1)2(1)2. The two domain fold found for OSBS is similar to those found for other members of the enolase superfamily: a mixed alpha/beta capping domain formed from segments at the N- and C-termini of the polypeptide and a larger (beta/alpha)(7)beta barrel domain. Two regions of disorder were found in the structure of apo OSBS: (i) the loop between the first two beta-strands in the alpha/beta domain; and (ii) the first sheet-helix pair in the barrel domain. These regions are ordered in the product complex with Mg(2+)*OSB. As expected, the Mg(2+)*OSB pair is bound at the C-terminal end of the barrel domain. The electron density for the phenyl succinate component of the product is well-defined; however, the 1-carboxylate appears to adopt multiple conformations. The metal is octahedrally coordinated by Asp(161), Glu(190), and Asp(213), two water molecules, and one oxygen of the benzoate carboxylate group of OSB. The loop between the first two beta-strands in the alpha/beta motif interacts with the aromatic ring of OSB. Lys(133) and Lys(235) are positioned to function as acid/base catalysts in the dehydration reaction. Few hydrogen bonding or electrostatic interactions are involved in the binding of OSB to the active site; instead, most of the interactions between OSB and the protein are either indirect via water molecules or via hydrophobic interactions. As a result, evolution of both the shape and the volume of the active site should be subject to few structural constraints. This would provide a structural strategy for the evolution of new catalytic activities in homologues of OSBS and a likely explanation for how the OSBS from Amycolaptosis also can catalyze the racemization of N-acylamino acids [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258].     

Menaquinone (Vitamin K2) Biosynthesis: Localization and Characterization of the menA Gene from Escherichia coli

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.     

Menaquinone biosynthesis: mutants of Escherichia coli K-12 requiring 2-succinylbenzoate.

Two independent mutants of Escherichia coli K-12, selected for their inability to grow anaerobically with fumarate as the terminal electron acceptor, were shown to be deficient in menaquinone biosynthesis. In both cases, exogenously supplied 2-succinylbenzoate promoted normal anaerobic growth on a lactate plus fumarate medium. Anaerobic growth of the mutants on glucose minimal medium was impaired but could be restored to normal by adding either uracil or 2-succinylbenzoate. The addition of 2-succinylbenzoate (but not uracil) permitted the synthesis of menaquinone and demethylmenaquinone by both mutants. The menaquinone content of the parental strain grown on lactate plus fumarate was three times greater than observed after growth on glucose. Transduction studies with phage P1 showed that the two mutations are very closely linked and probably affect the same gene, menC, which is cotransducible with nalA (23%), glpT (51%), and purF (8 to 14%). The gene order nalA-nrdA-glpTA-menC-purF was indicated. The results were consistent with 2-succinylbenzoate being an intermediate in menaquinone biosynthesis and show that the gene designated menC (located at 48.65 min of the E. coli chromosome) is involved in the conversion of chorismate to 2-succinylbenzoate. It was also concluded that menaquinone is essential for electron transport to fumarate in E. coli.     

Vitamin B12 transport in Escherichia coli K12 does not require the btuE gene of the btuCED operon

Summary Transport of vitamin B₁₂ across the cytoplamic membrane ofEscherichia coli requires the products ofbtuC andbtuD, two genes in thebtuCED operon. The role ofbtuE, the central gene of this operon, was examined. Deletions withinbtuE were constructed by removal of internal restriction fragments and were crossed onto the chromosome by allelic replacement. In-frame deletions that removed 20% or 82% of thebtuE coding region did not affect expression of the distalbtuD gene. These nonpolar deletions had little effect on vitamin B₁₂ binding (whole cells or periplasmic fraction) and transport. They did not affect the utilization of vitamin B₁₂ or other cobalamins for methionine biosynthesis, even in strains with decreased outer membrane transport of vitamin B₁₂. ThebtuE mutations did not impair adenosyl-cobalamin dependent catabolism of ethanolamine or repression ofbtuB expression. Thus, despite its genetic location in the transport operon, thebtuE product plays no essential role in vitamin B₁₂ transport.     

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

In Escherichia coli, isochorismate is a common precursor for the biosynthesis of the siderophore enterobactin and menaquinone (vitamin K2). Isochorismate is formed by the shikimate pathway from chorismate by the enzyme isochorismate synthase encoded by the entC gene. Since enterobactin is involved in the aerobic assimilation of iron, and menaquinone is involved in anaerobic electron transport, we investigated the regulation of entC by iron and oxygen. An operon fusion between entC with its associated regulatory region and lacZ+ was constructed and introduced into the chromosome in a single copy. Expression of entC-lacZ was found to be regulated by the concentration of iron both aerobically and anaerobically. An established entC::kan mutant deficient in enterobactin biosynthesis was found to grow normally and synthesize wild-type levels of menaquinone under anaerobic conditions in iron-sufficient media. These results led to the demonstration of an alternate isochorismate synthase specifically involved in menaquinone synthesis encoded by the menF gene. Consistent with these findings, the entC+ strains were found to synthesize enterobactin anaerobically under iron-deficient conditions while the ent mutants failed to do so.     

Outer membrane translocation of the extracellular enzyme pullulanase in Escherichia coli K12 does not require a fatty acylated N-terminal cysteine.

Site-directed mutagenesis was used to construct three mutant derivatives of the extracellular, cell surface lipoprotein pullulanase (PulA) in which the normally fatty acylated cysteine of the signal peptide-bearing precursor was replaced by other amino acids. When produced in Escherichia coli expressing all genes required for pullulanase secretion, approximately 90% of the PulA derivatives persisted as cell-associated precursors, indicating inefficient signal peptide processing. Processed (intermediate-sized) forms of the two derivatives that were studied in detail were found to result from proteolytic cleavage at different sites within the signal peptide. Both were further processed to smaller polypeptides by cleavage at an undetermined site that is presumably close to their C termini. The intermediate-sized pullulanase derived from prepullulanase in which Cys+1 had been replaced by Leu and Gly-1 by Glu (PulA:C1L/G-1E) appeared rapidly, was apparently entirely extracellular, and accounted for approximately 10% of synthesized PulA. Prolonged incubation did not result in further conversion of the precursor to the intermediate form, and the precursor remained anchored to the cytoplasmic membrane. The smaller processed form was also found extracellularly. The active form of the extracellular enzyme was monomeric, which is again in contrast to the fatty acylated, wild-type enzyme. Taken together, these results indicate that replacement of Cys+1 of prePulA eliminates processing by lipoprotein signal peptidase and does not permit processing by leader peptidase, but allows inefficient, aberrant processing by an unknown peptidase and immediate secretion of the resulting polypeptide, which retains most of its signal peptide. Processing and secretion only occur when the pullulanase secretion functions are expressed.