Antibacterial activity of 2-amino-4-hydroxypyrimidine-5-carboxylates and binding to Burkholderia pseudomallei 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase.

Enzymes in the methylerythritol phosphate pathway make attractive targets for antibacterial activity due to their importance in isoprenoid biosynthesis and the absence of the pathway in mammals. The fifth enzyme in the pathway, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), contains a catalytically important zinc ion in the active site. A series of de novo designed compounds containing a zinc binding group was synthesized and evaluated for antibacterial activity and interaction with IspF from Burkholderia pseudomallei, the causative agent of Whitmore's disease. The series demonstrated antibacterial activity as well as protein stabilization in fluorescence-based thermal shift assays. Finally, the binding of one compound to Burkholderia pseudomallei IspF was evaluated through group epitope mapping by saturation transfer difference NMR.

Synthesis and biological evaluation of pyrazolopyrimidines as potential antibacterial agents.

The fragment FOL7185 (compound 17) was found to be a hit against IspD and IspE enzymes isolated from bacteria, and a series of analogs containing the pyrazolopyrimidine core were synthesized. The majority of these compounds inhibited the growth of Burkholderia thailandensis (Bt) and Pseudomonas aeruginosa (Pa) in the Kirby­Bauer disk diffusion susceptibility test. Compound 29 shows inhibitory activity at 0.1 mM (32.2 lg/mL), which is comparable to the control compound kanamycin (48.5 lg/mL). Compound 29 also shows inhibitory activity at 0.5 mM against kanamycin resistant P. aeruginosa. Saturation transfer difference NMR (STD-NMR) screening of these compounds against BtIspD and BtIspE indicated that most of these compounds significantly interact with BtIspE, suggesting that the compounds may inhibit the growth of Bt by disrupting isoprenoid biosynthesis. Ligand epitope mapping of compound 29 with BtIspE indicated that hydrogens on 2,4-dichlorophenyl group have higher proximity to the surface of the enzyme than hydrogens on the pyrazolopyrimidine ring.

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 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.

Ubiquinone biosynthesis in microorganisms.

The quinoid nucleus of the benzoquinone, ubiquinone (coenzyme Q; Q), is derived from the shikimate pathway in bacteria and eukaryotic microorganisms. Ubiquinone is not considered a vitamin since mammals synthesize it from the essential amino acid tyrosine. Escherichia coli and other Gram-negative bacteria derive the 4-hydroxybenzoate required for the biosynthesis of Q directly from chorismate. The yeast, Saccharomyces cerevisiae, can either form 4-hydroxybenzoate from chorismate or tyrosine. However, unlike mammals, S. cerevisiae synthesizes tyrosine in vivo by the shikimate pathway. While the reactions of the pathway leading from 4-hydroxybenzoate to Q are the same in both organisms the order in which they occur differs. The 4-hydroxybenzoate undergoes a prenylation, a decarboxylation and three hydroxylations alternating with three methylation reactions, resulting in the formation of Q. The methyl groups for the methylation reactions are derived from S-adenosylmethionine. While the prenyl side chain is formed by the 2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway in E. coli, it is formed by the mevalonate pathway in the yeast.

Dimethyl sulfoxide reductase is not required for trimethylamine N-oxide reduction in Escherichia coli.

Deletion mutants of Escherichia coli lacking dimethyl sulfoxide (DMSO) reductase activity and consequently unable to utilize DMSO as an electron acceptor for anaerobic growth have been isolated. These mutants retained the ability to use trimethylamine N-oxide (TMAO) as an electron acceptor and the TMAO reductase activity was found to be unaltered. Heating the cell-free extract of the wild-type strain at 70 degrees C for 15 min selectively inactivated the DMSO reductase activity while the TMAO reductase activity remained unchanged for at least 1 h.

Ubiquinone (coenzyme Q) biosynthesis in Escherichia coli: identification of the ubiF gene.

Ubiquinone (coenzyme Q; abbreviation, Q) plays an essential role in electron transport in Escherichia coli when oxygen or nitrate is the electron acceptor. The biosynthesis of Q involves at least nine reactions. Three of these reactions involve hydroxylations resulting in the introduction of hydroxyl groups at positions C-6, C-4, and C-5 of the benzene nucleus of Q. The genes encoding the enzymes responsible for these hydroxylations, ubiB, ubiH, and ubiF are located at 87, 66, and 15 min of the E. coli linkage map. The ubiF encoded oxygenase introduces the hydroxyl group at carbon five of 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol resulting in the formation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1, 4-benzoquinol. An ubiF mutant failed to carry out this conversion. Based on the homology to UbiH, an open reading frame (orf391) was identified at the 15 min region of the chromosome, amplified using PCR, and cloned into pUC18 plasmid. The ubiF mutants, when complemented with this plasmid, regained the ability to grow on succinate and synthesize Q.

A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene.

A new gene (menF) encoding an isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis has been cloned and sequenced. Overexpression of the encoded polypeptide under the influence of a T7 promoter showed an increase in specific activity of 2200-fold. Treatment with protamine sulfate resulted in another 3.5-fold increase in specific activity (7700-fold compared to the parent strain). The relative molecular mass of the overexpressed protein was M(r) 49 000, which is in full agreement with the DNA sequence predicted molecular mass of 48 777 Da. Purified enzyme converted chorismate to isochorismate with the product of the reaction shown to be isochorismate by its thermal conversion to salicylic acid. The fluorescence spectrum generated by the formed salicylic acid was identical to that of authentic salicylic acid. The 5' end of the flanking menD gene has also be redefined.

Vitamin K2 is a mitochondrial electron carrier that rescues pink1 deficiency.

Human UBIAD1 localizes to mitochondria and converts vitamin K(1) to vitamin K(2). Vitamin K(2) is best known as a cofactor in blood coagulation, but in bacteria it is a membrane-bound electron carrier. Whether vitamin K(2) exerts a similar carrier function in eukaryotic cells is unknown. We identified Drosophila UBIAD1/Heix as a modifier of pink1, a gene mutated in Parkinson's disease that affects mitochondrial function. We found that vitamin K(2) was necessary and sufficient to transfer electrons in Drosophila mitochondria. Heix mutants showed severe mitochondrial defects that were rescued by vitamin K(2), and, similar to ubiquinone, vitamin K(2) transferred electrons in Drosophila mitochondria, resulting in more efficient adenosine triphosphate (ATP) production. Thus, mitochondrial dysfunction was rescued by vitamin K(2) that serves as a mitochondrial electron carrier, helping to maintain normal ATP production.

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

Escherichia coli and Salmonella contain the naphthoquinones menaquinone (MK; vitamin K2) and demethylmenaquinone and the benzoquinone ubiquinone (coenzyme Q; Q). Both quinones are derived from the shikimate pathway, which has been called a "metabolic tree with many branches." There are two different pathways for the biosynthesis of the naphthoquinones. The vast majority of prokaryotes, including E. coli and Salmonella, and the plants use the o-succinylbenzoate pathway, while a minority uses the futalosine pathway. The quinone nucleus of Q is derived directly from chorismate, while that of MK is derived from chorismate via isochorismate. The prenyl side chains of both quinones are from isopentenyl diphosphate formed by the 2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway and the methyl groups are from S-adenosylmethionine. In addition, MK biosynthesis requires 2-ketoglutarate and cofactors ATP, coenzyme A, and thiamine pyrophosphate. Despite the fact that both quinones originate from the shikimate pathway, there are important differences in their biosyntheses. The prenyl side chain in MK biosynthesis is introduced at the penultimate step, accompanied by decarboxylation, whereas in Q biosynthesis it is introduced at the second step, with retention of the carboxyl group. In MK biosynthesis, all the reactions of the pathway up to prenylation are carried out by soluble enzymes, whereas all the enzymes involved in Q biosynthesis except the first are membrane bound. In MK biosynthesis, the last step is a C-methylation; in Q biosynthesis, the last step is an O-methylation. In Q biosynthesis a second C-methylation and O-methylation take place in the middle part of the pathway. Despite the fact that Q and MK biosyntheses diverge at chorismate, the C-methylations in both pathways are carried out by the same methyltransferase.

Regulation of the ubiquinone (coenzyme Q) biosynthetic genes ubiCA in Escherichia coli.

Ubiquinone (Coenzyme Q) is an essential component of bacterial respiratory chains. The first committed step in the biosynthetic pathway is the formation of 4-hydroxybenzoate from chorismate by the enzyme chorismate pyruvate-lyase encoded by the ubiC gene. The 4-hydroxybenzoate is prenylated by 4-hydroxybenzoate octaprenyltransferase encoded by the ubiA gene. The two genes are linked at 91.5 min in the Escherichia coli chromosome. To study the regulation, operon fusions were constructed between these two genes and the lacZ gene. The fusions were introduced into the chromosome as a single copy at the lambda attachment site. Expression of beta-galactosidase was determined in strains carrying the operon fusions ubiC'-lacZ(+) ubiCA'-lacZ(+), and ubiA'-lacZ(+). In glycerol media, the highest level of expression was observed with the operon fusion ubiC'-lacZ(+). Compared with the ubiC'-lacZ(+), the ubiCA'-lacZ(+) operon fusion showed 26% of the activity while the ubiA'-lacZ(+) operon fusion had an activity of 1%. Thus, the ubiC gene is regulated by the upstream promoter while the ubiA gene lacks its own promoter. The effect of fermentable and oxidizable carbon sources on the expression of ubiC'-lacZ(+) was determined. The expression was low in the case of a fermentable carbon source, glucose, while in the presence of oxidizable carbon sources the expression increased 2- to 3-fold. When the expression of ubiC'-lacZ(+) and ubiCA'-lacZ(+) operon fusions were compared under a wide variety of conditions, the levels of beta-galactosidase varied coordinately, suggesting that the ubiCA genes are organized into an operon. The variations in transcription of the operon under different nutritional conditions and in the regulatory mutants, arcA, fnr, and narXL are presented.

Enzymes from Escherichia coli synthesize o-succinylbenzoic acid, an intermediate in menaquinone (vitamin K2) biosynthesis.

Cell-free preparations have been obtained from Escherichia coli AN 154 which catalyze the conversion of chorismic acid and alpha-ketoglutaric acid to o-succinylbenzoic acid. This result constitutes the first experimental verification of the committed step in menaquinone biosynthesis. The enzymatic biosynthesis of o-succinylbenzoic acid was demonstrated in experiments utilizing [U-14C]alpha-ketoglutaric acid as substrate. Following purification of O-succinylbenzoic acid and conversion to the dimethyl derivative, the presence of 14C was shown by scanning of thin layer chromatograms, and by radiogas chromatography. Proof for the formation of o-succinylbenzoic acid was also obtained by combined gas chromatography/mass spectrometry. The formation of o-succinylbenzoic acid in these extracts required the presence of thiamin pyrophosphate; when this cofactor was omitted from the incubations, there was a substantial diminution in the incorporation of radioactivity from alpha-ketoglutarate into o-succinylbenzoic acid (from 4- to 8-fold). These results support the suggestion (Campbell, I. M. (1969) Tetrahedron Lett. 4777-4780) that the three-carbon unit of lawsone and, hence by inference, the four-carbon side chain of o-succinylbenzoic acid, is derived from the thiamin pyrophosphate adduct of succinic semialdehyde (likely in the anion form). The activated form of succinic semialdehyde is derived by the action of the first enzyme of the alpha-ketoglutarate dehydrogenase complex or by a similar decarboxylase enzyme.

Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): a perspective on enzymatic mechanisms.

The benzoquinone ubiquinone (coenzyme Q) and the naphthoquinones menaquinone (vitamin K2) and demethylmenaquinone are derived from the shikimate pathway, which has been described as a "metabolic tree with many branches." Menaquinone (MK) is considered a vitamin, but coenzyme (Q) is not; MK is an essential nutrient (it cannot be synthesized by mammals), whereas Q is not considered an essential nutrient since it can be synthesized from the amino acid tyrosine. The quinone nucleus of Q is derived directly from chorismate, whereas that of MK is derived from chorismate via isochorismate. The prenyl side chain of both quinones is derived from prenyl diphosphate, and the methyl groups are derived from S-adenosylmethionine. MK biosynthesis requires 2-ketoglutarate and the cofactors ATP, coenzyme A (CoASH), and thiamine pyrophosphate. In spite of the fact that both quinones originate from the shikimate pathway, there are important differences in their biosynthesis. In MK biosynthesis, the prenyl side chain is introduced in the next to last step, which is accompanied by loss of the carboxyl group, whereas in Q biosynthesis, the prenyl side chain is introduced at the second step, with retention of the carboxyl group. In MK biosynthesis, all the reactions of the pathway up to the prenylation (next to last step) are carried out by soluble enzymes, whereas all the enzymes involved in Q biosynthesis except the first are membrane bound. In MK biosynthesis the last step is a C-methylation; in Q biosynthesis, the last step is an O-methylation. In Q biosynthesis a second C-methylation and O-methylation take place in the middle part of the pathway. In spite of the fact that Q and MK biosynthesis diverges at chorismate, the C-methylations involved in both pathways are carried out by the same enzyme. Finally, Q biosynthesis under aerobic conditions requires molecular oxygen; anaerobic biosynthesis of Q and MK incorporates oxygen atoms derived from water. The current status of the pathways with particular emphasis on the reaction mechanisms, is discussed in this review.

Tetrahydrothiophene 1-oxide as an electron acceptor for Escherichia coli.

Escherichia coli used tetrahydrothiophene 1-oxide (THTO) as an electron acceptor for anaerobic growth with glycerol as a carbon source; the THTO was reduced to tetrahydrothiophene. Cell extracts also reduced THTO to tetrahydrothiophene in the presence of a variety of electron donors. Chlorate-resistant (chl) mutants (chlA, chlB, chlD, and chlE) were unable to grow with THTO as the electron acceptor. However, growth and THTO reduction by the chlD mutant were restored by high concentrations of molybdate. Similarly, mutants of E. coli that are blocked in the menaquinone (vitamin K2) biosynthetic pathway, i.e., menB, menC, and menD mutants, did not grow with THTO as an electron acceptor. Growth and THTO reduction were restored in these mutants by the presence of appropriate intermediates of the vitamin K biosynthetic pathway.

Dimethyl sulphoxide respiration in Proteus mirabilis.

Dimethyl sulphoxide (DMSO) was found to serve as an electron acceptor for the anaerobic growth of Proteus mirabilis on fermentable substrates such as glucose and pyruvate, as well as on oxidizable substrates such as glycerol and lactate. In a complex medium, formate greatly stimulated growth in the presence of DMSO. Cell extracts were found to reduce DMSO to dimethyl sulphide (DMS) in the presence of an electron donor. It was found that NADH, formate, lactate, reduced benzyl viologen, and dithionite can serve as electron donors. Chlorate resistant (chl) mutants were found to be unable to grow using DMSO as an electron acceptor. However, in one chl mutant, growth and DMSO reduction could be partially restored by growth in the presence of high concentrations of molybdate.

Thiamine pyrophosphate requirement for o-succinylbenzoic acid synthesis in Escherichia coli and evidence for an intermediate.

Cell-free extracts of various strains of Escherichia coli synthesize the menaquinone biosynthetic intermediate o-succinylbenzoic acid (OSB) when supplied with chorismic acid, 2-ketoglutaric acid, and thiamine pyrophosphate (TPP). To assay for OSB synthesis, 2-[U-14C]ketoglutaric acid was used as substrate, and the synthesized OSB was examined by radiogas chromatography (as the dimethyl ester). [U-14C]Shikimic acid also gave rise to radioactive OSB if the cofactors necessary for enzymatic conversion to chorismic acid were added. Use of 2-[1-14C]ketoglutaric acid does not give rise to labeled OSB. In the absence of TPP during the incubations, OSB synthesis was much reduced; these observations are consistent with the proposed role for the succinic semialdehyde-TPP anion as the reagent adding to chorismic acid. Extracts of cells from menC and menD mutants did not form OSB separately, but did so in combination. There was evidence for formation of a product, X, by extracts of a menC mutant incubated with chorismic acid, TPP, and 2-ketoglutaric acid; X was converted to OSB by extracts of a menD mutant. It appears that the intermediate, X, is formed by one gene product and converted to OSB by the second gene product.

Menaquinone (vitamin K2) biosynthesis: conversion of o-succinylbenzoic acid to 1,4-dihydroxy-2-naphthoic acid by Mycobacterium phlei enzymes.

The coenzyme A (CoA) and adenosine 5'-triphosphate-dependent conversion of o-succinylbenzoic acid (4-[2'-carboxyphenyl]-4-oxobutyric acid) to 1,4-dihydroxy-2-naphthoic acids is an important step in menaquinone (vitamin K2) biosynthesis. Cell-free extracts catalyzing this conversion, obtained from Mycobacterium phlei, were separated into three protein fractions by treatment with protamine sulfate. The second fraction (fraction B) and the supernatant (fraction S) alone did not catalyze dihydroxynaphthoate formation, but did so in combination. All of the results were consistent with the formation of an unstable intermediate, likely an o-succinylbenzoyl-CoA compound, by the action of fraction S. Adenosine 5'-triphosphate was required in this reaction and adenosine 5'-monophosphate was formed. This enzyme activity was termed o-succinylbenzoyl-CoA synthetase: the enzyme showed a marked stability to 0.1 N hydrochloric acid. The presumed o-succinylbenzoyl-CoA derivate was rather unstable; under a variety of conditions, it was converted to a spirodilactone form of o-succinylbenzoate. Fraction B contained an enzyme, termed naphthoate synthase, which converted the o-succinylbenzoyl-CoA derivative to 1,4-dihydroxy-2-naphthoate.

Biosynthesis of o-succinylbenzoic acid in a men- Escherichia coli mutant requires decarboxylation of L-glutamate at the C-1 position.

A men- mutant of Escherichia coli, AN 209, which accumulates o-succinylbenzoic acid, has been used for a direct study of the biosynthesis of this benzenoid compound. Samples of labeled glutamic acids were added to growth media, and the o-succinylbenzoic acid was isolated and converted to a dimethyl derivative. This dimethyl derivative was purified on thin-layer chromatograms and by gas chromatography. When the glutamic acid used as precursor contained 14C at position 5, or was uniformly labeled, the dimethyl o-succinylbenzoate contained radioactivity (as shown by radiogas chromatography). However, from [1-14C]glutamate, the dimethyl o-succinylbenzoate was without radioactivity. Hence, in the biosynthesis of o-succinylbenzoate, carbon atom 1 of glutamate is lost, and carbon atoms 2-5 are retained. It was also shown that this mutant lacked the enzyme dihydroxynaphthoic acid synthase. It should, therefore, continue to be classified as a menB mutant, rather than as a member of the newly created menE group (lacking o-succinylbenzoate-CoA synthetase).

Identity of the quinone in Bacillus alcalophilus.

Every Bacillus species so far examined contains menaquinone as the sole quinone. In contrast, the alkalophilic Bacillus alcalophilus has been reported to be unusual in containing ubiquinone rather than menaquinone. In this communication, we demonstrate that B. alcalophilus, like all the other bacilli, contains menaquinone as the only quinone.

Identification of Bacillus subtilis men mutants which lack O-succinylbenzoyl-coenzyme A synthetase and dihydroxynaphthoate synthase.

Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis, whose growth requirement is satisfied by 1,4-dihydroxy-2-naphthoic acid but not by o-succinylbenzoic acid (OSB), have been analyzed for enzymatic defects. Complementation analysis of cell-free extracts of the mutants revealed that there are two groups, as already indicated by genetic analysis. The missing enzyme in each group was identified by complementation of the cell-free extracts with o-succinylbenzoyl-coenzyme A (CoA) synthetase and dihydroxynaphthoate synthase extracted from Mycobacterium phlei. Mutants found to lack dihydroxynaphthoate synthase, and which therefore complement with dihydroxynaphthoate synthase of M. phlei, were designated as menB; those lacking o-succinylbenzoyl-CoA synthetase, and therefore complementing with o-succinylbenzoyl-CoA synthetase, were designated as menE. The menB mutants RB413 (men-325) and RB415 (men-329), when incubated with [2,3-14C2]OSB, produced only the spirodilactone form of OSB in a reaction that was CoA and adenosine 5'-triphosphate dependent.