Bacterial phenylalanine and phenylacetate catabolic pathway revealed.

Aromatic compounds constitute the second most abundant class of organic substrates and environmental pollutants, a substantial part of which (e.g., phenylalanine or styrene) is metabolized by bacteria via phenylacetate. Surprisingly, the bacterial catabolism of phenylalanine and phenylacetate remained an unsolved problem. Although a phenylacetate metabolic gene cluster had been identified, the underlying biochemistry remained largely unknown. Here we elucidate the catabolic pathway functioning in 16% of all bacteria whose genome has been sequenced, including Escherichia coli and Pseudomonas putida. This strategy is exceptional in several aspects. Intermediates are processed as CoA thioesters, and the aromatic ring of phenylacetyl-CoA becomes activated to a ring 1,2-epoxide by a distinct multicomponent oxygenase. The reactive nonaromatic epoxide is isomerized to a seven-member O-heterocyclic enol ether, an oxepin. This isomerization is followed by hydrolytic ring cleavage and beta-oxidation steps, leading to acetyl-CoA and succinyl-CoA. This widespread paradigm differs significantly from the established chemistry of aerobic aromatic catabolism, thus widening our view of how organisms exploit such inert substrates. It provides insight into the natural remediation of man-made environmental contaminants such as styrene. Furthermore, this pathway occurs in various pathogens, where its reactive early intermediates may contribute to virulence.

Analysis of a chemical plant defense mechanism in grasses.

In the Gramineae, the cyclic hydroxamic acids 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one (DIMBOA) form part of the defense against insects and microbial pathogens. Five genes, Bx1 through Bx5, are required for DIBOA biosynthesis in maize. The functions of these five genes, clustered on chromosome 4, were demonstrated in vitro. Bx1 encodes a tryptophan synthase alpha homolog that catalyzes the formation of indole for the production of secondary metabolites rather than tryptophan, thereby defining the branch point from primary to secondary metabolism. Bx2 through Bx5 encode cytochrome P450-dependent monooxygenases that catalyze four consecutive hydroxylations and one ring expansion to form the highly oxidized DIBOA.

Deoxyxylulose phosphate pathway to terpenoids.

Recently, a mevalonate-independent pathway was discovered in bacteria and plants that leads to the formation of isopentenyl diphosphate and dimethylallyl diphosphate, the two basic precursors of isoprenoids. Although many details of the widely distributed pathway are unknown, some intermediates, mechanisms, enzymes and genes of this novel route have been identified. Information on this pathway could provide the basis for the development of new antibiotics, herbicides and antimalarials.

The non-mevalonate pathway of isoprenoids: genes, enzymes and intermediates.

Although the mevalonate pathway had been considered for a long time as the unique source of biosynthetic isoprenoids, an alternative pathway has recently been discovered. The first intermediate, 1-deoxy-D-xylulose 5-phosphate, is assembled by condensation of glyceraldehyde 3-phosphate and pyruvate. A skeletal rearrangement coupled with a reduction step affords the branched-chain polyol, 2C-methyl-D-erythritol 4-phosphate, which is subsequently converted into a cyclic 2,4-diphosphate by the consecutive action of three enzymes via nucleotide diphosphate intermediates. The genes specifying these enzymes have been cloned from bacteria, plants and protozoa. Their expression in recombinant bacterial hosts has opened the way to the identification of several novel pathway intermediates.

The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms.

Recent studies have uncovered the existence of an alternative, non-mevalonate pathway for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate, the two building blocks of terpene biosynthesis.

Biosynthesis of terpenoids: 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Escherichia coli is a class B dehydrogenase.

1-Deoxy-D-xylulose-5-phosphate is converted into 2-C-methyl-D-erythritol-4-phosphate by the catalytic action of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr protein) using NADPH as cofactor. The stereochemical features of this reaction were investigated in in vitro experiments with the recombinant Dxr protein of Escherichia coli using (4R)- or (4S)-[4-(2)H(1)]NADPH as coenzyme. The enzymatically formed 2-C-methyl-D-erythritol-4-phosphate was isolated and converted into 1,2:3,4-di-O-isopropylidene-2-C-methyl-D-erythritol; NMR spectroscopic investigation of this derivative indicated that only (4S)-[4-(2)H(1)]NADPH affords 2-C-methyl-D-erythritol-4-phosphate labelled exclusively in the H(Re) position of C-1. Stereospecific transfer of H(Si) from C-4 of the cofactor identifies the Dxr protein of E. coli as a class B dehydrogenase.

Biosynthesis of thiophenes in Tagetes patula.

The biosynthesis of 5-(3-buten-1-ynyl)-2,2'-bithiophene was studied in root cultures of Tagetes patula. Organ cultures were grown with [U-13C(6)]glucose or [1-13C]glucose. The bithiopene and amino acids from cell protein were isolated and analysed by quantitative NMR spectroscopy. Retrobiosynthetic analysis establish acetyl-CoA or a closely related compound (e.g. malonyl-CoA) as building blocks and their orientations in the bithiophene. The data confirm a previously suggested biosynthetic route via long-chain fatty acids and polyacetylenes. However, a polyketide-like biosynthesis via a carbocyclic intermediate cannot be excluded.

Tracer studies with 13C-labeled carbohydrates in cultured plant cells. Retrobiosynthetic analysis of chelidonic acid biosynthesis.

The biosynthesis of chelidonic acid was studied in cell suspension cultures of Leucojum aestivum. Cell cultures were supplied with [U-13C]glucose, [l-13C]glucose or [U-13Cs]ribose/ribulose in standard medium containing unlabeled glucose. 13C labeling patterns of amino acids obtained by hydrolysis of biomass were determined by NMR spectroscopy and compared to the labeling pattern of chelidonic acid. The data document the incorporation of a contiguous 4-carbon fragment derived from the pentose phosphate pool into chelidonic acid. This suggests a biosynthetic pathway involving the condensation of phosphoenolpyruvate with a pentose phosphate followed by dehydration, dehydrogenation, ring closure and decarboxylation conducive to the loss of C-5 of the pentose precursor.

Synthesis and analysis of thio-, thiono-, and dithio-derivatives of whiskey lactone.

Cis- and trans-3-methyl-4-octanolide (1, whiskey lactones) were converted into their thio- (2), thiono- (3), and dithio- (4) derivatives by reaction with phosphorus pentasulfide. The reaction products were characterized by GC-mass spectrometry, (1)H NMR spectroscopy, and GC-olfactometry. Two-dimensional NOESY spectra showed that sulfur is incorporated into the ring with reversal of the absolute configuration at C-4, whereas substitution of the keto-oxygen atom by sulfur occurs with retention of ring configuration. The cis- and trans-pairs of 2, 3, and 4 were separated into enantiomers by GC on heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin and heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin as chiral stationary phases. GC-olfactometry revealed a sweet coconut-like odor for the cis-thio- and pleasant mushroom-like flavors for the cis-thiono- and trans-dithio-derivatives of whiskey lactone.

Anti-malarial drug targets: screening for inhibitors of 2C-methyl-D-erythritol 4-phosphate synthase (IspC protein) in Mediterranean plants.

The recently discovered non-mevalonate pathway of isoprenoid biosynthesis serves as the unique source of terpenoids in numerous pathogenic eubacteria and in apicoplast-type protozoa, most notably Plasmodium, but is absent in mammalian cells. It is therefore an attractive target for anti-infective chemotherapy. The first committed step of the non-mevalonate pathway is catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase (IspC). Using photometric and NMR spectroscopic assays, we screened extracts of Mediterranean plants for inhibitors of the enzyme. Strongest inhibitory activity was found in leaf extracts of Cercis siliquastrum.

Isoprenoid biosynthetic pathways as anti-infective drug targets.

IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) serve as the universal precursors for the biosynthesis of isoprenoids. Besides the well-known mevalonate pathway, the existence of a second biosynthetic pathway conducive to IPP and DMAPP formation through 1-deoxy-D-xylulose 5-phosphate and 2C-methyl-D-erythritol 4-phosphate was discovered approx. 10 years ago in plants and certain eubacteria. It is now known that this pathway is widely distributed in the bacterial kingdom including major human pathogens, such as Mycobacterium tuberculosis and Helicobacter pylori. The pathway is also essential in the malaria vector Plasmodium falciparum. During the last few years, the genes, enzymes, intermediates and mechanisms of the biosynthetic route have been elucidated by a combination of comparative genomics, enzymology, advanced NMR technology and crystallography. The results provide the basis for the development of novel anti-infective drugs.

Structures and reaction mechanisms of riboflavin synthases of eubacterial and archaeal origin.

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. GTP is hydrolytically opened, converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate leads to 6,7-dimethyl-8-ribityllumazine. The dismutation of 6,7-dimethyl-8-ribityllumazine catalysed by riboflavin synthase produces riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. A pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazines has been identified earlier as a catalytically competent reaction intermediate of the Escherichia coli enzyme. Acid quenching of reaction mixtures of riboflavin synthase of Methanococcus jannaschii, devoid of similarity to riboflavin synthases of eubacteria and eukaryotes, afforded a compound whose optical absorption and NMR spectra resemble that of the pentacyclic E. coli riboflavin synthase intermediate, whereas the CD spectra of the two compounds have similar envelopes but opposite signs. Each of the compounds could serve as a catalytically competent intermediate for the enzyme by which it was produced, but not vice versa. All available data indicate that the respective pentacyclic intermediates of the M. jannaschii and E. coli enzymes are diastereomers. Whereas the riboflavin synthase of M. jannaschii is devoid of similarity with those of eubacteria and eukaryotes, it has significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalysing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor.

Biosynthesis of methanofuran in Methanobacterium thermoautotrophicum.

The 13C NMR signals of methanofuran were assigned by two-dimensional 1H and 13C NMR experiments. On this basis, the incorporation of 13C-labeled acetate and pyruvate into methanofuran by growing cells of Methanobacterium thermoautotrophicum was analyzed by one- and two-dimensional 13C NMR experiments. The data were analyzed by a retrobiosynthetic approach based on a comparison of labeling patterns in a variety of metabolites. The data show that the furan ring is formed by condensation of two molecules from the pyruvate/triose pool. The tetracarbocylic acid moiety is assembled from ketoglutarate, two molecules of acetyl CoA, and one molecule of carbon dioxide.

Biosynthesis of 5-hydroxybenzimidazolylcobamid (factor III) in Methanobacterium thermoautotrophicum.

Cultures of Methanobacterium thermoautotrophicum were supplemented with 13C-labeled acetate or pyruvate, and the labeling pattern of the corrinoid, factor III, was established by 13C NMR spectroscopy. Complete 13C signal assignments were obtained by two-dimensional NMR experiments. The labeling pattern of factor III was analyzed by comparison with those of amino acids and nucleosides. The corrin ring system is derived from eight molecules of glutamate. The aminopropanol moiety is derived in a hitherto unknown pathway from pyruvate by reductive amination. The heterocyclic ring of hydroxybenzimidazole shares the labeling pattern of the imidazole ring of purines. The remaining four carbon atoms of the carbocyclic ring show the labeling signature of a carbohydrate with two of the carbons introduced from acetate and two from C-1 of pyruvate. However, erythrose can be ruled out as the specific precursor on the basis of a detailed investigation of aromatic amino acids indicating that erythrose 4-phosphate is obtained by reductive carboxylation of a triose precursor and not by the pentose phosphate cycle.

A pentacyclic reaction intermediate of riboflavin synthase.

The S41A mutant of riboflavin synthase from Escherichia coli catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a very low rate. Quenching of presteady-state reaction mixtures with trifluoroacetic acid afforded a compound with an absorption maximum at 412 nm (pH 1.0) that can be converted to a mixture of riboflavin and 6,7-dimethyl-8-ribityllumazine by treatment with wild-type riboflavin synthase. The compound was shown to qualify as a kinetically competent intermediate of the riboflavin synthase-catalyzed reaction. Multinuclear NMR spectroscopy, using various 13C- and 15N-labeled samples, revealed a pentacyclic structure arising by dimerization of 6,7-dimethyl-8-ribityllumazine. Enzyme-catalyzed fragmentation of this compound under formation of riboflavin can occur easily by a sequence of two elimination reactions.

Crotoflavol, a new phenanthrene from Croton flavens.

Phytochemical investigations of the leaves of Croton flavens L. var. balsamiferus (Jacq.) Muell. Arg. has led to the isolation of the novel 2,5-dihydroxy-3,6-dimethoxyphenanthrene crotoflavol. The structure was elucidated on the basis of spectroscopic evidence. This is the first report on the isolation of a phenanthrene from a Croton species.

Biosynthesis of nucleotides, flavins, and deazaflavins in Methanobacterium thermoautotrophicum.

The biosynthesis of deazaflavins, flavins, ribonucleotides, and selected amino acids was studied in Methanobacterium thermoautotrophicum by incorporation of 13C-labeled acetate and pyruvate. 13C enrichments were monitored by 13C and 1H NMR spectroscopy. The biosynthesis of ribonucleotides follows the standard pathway. The xylene ring of riboflavin is formed from two pentose moieties in agreement with studies in yeasts and eubacteria. The pyrimidine ring and the ribityl side chain of the deazaflavin chromophore of coenzyme F420 are derived from the purine nucleotide pool. The phenolic ring and C-5 of the deazaflavin system are supplied by the shikimate pathway. A hypothetical mechanism for the assembly of the deazaflavin chromophore from 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione and 4-hydroxyphenyl-pyruvate is proposed.