Phosphopantothenoylcysteine synthetase from Escherichia coli. Identification and characterization of the last unidentified coenzyme A biosynthetic enzyme in bacteria

Phosphopantothenoylcysteine synthase catalyzes the formation of (R)-4'-phospho-N-pantothenoylcysteine from 4'-phosphopantothenate and l-cysteine: this enzyme, involved in the biosynthesis of coenzyme A (CoA), has not previously been identified. Recently it was shown that the NH(2)-terminal domain of the Dfp protein from bacteria catalyzes the next step in CoA biosynthesis, the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to form 4'-phosphopantetheine (Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M., and Steinbacher, S. (2000) J. Biol. Chem. 275, 31838-31846). We have partially purified phosphopantothenoylcysteine decarboxylase from Escherichia coli and demonstrated that the protein encoded by the dfp gene, here renamed coaBC, also has phosphopantothenoylcysteine synthetase activity, using CTP rather than ATP as the activating nucleoside 5'-triphosphate. This discovery completes the identification of all the enzymes involved in the biosynthesis of coenzyme A in bacteria.     

Holo-(acyl carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli

Holo-(acyl carrier protein) synthase (AcpS) post-translationally modifies apoacyl carrier protein (apoACP) via transfer of 4'-phosphopantetheine from coenzyme A (CoA) to the conserved serine 36 gamma-OH of apoACP. The resulting holo-acyl carrier protein (holo-ACP) is then active as the central coenzyme of fatty acid biosynthesis. The acpS gene has previously been identified and shown to be essential for Escherichia coli growth. Earlier mutagenic studies isolated the E. coli MP4 strain, whose elevated growth requirement for CoA was ascribed to a deficiency in holoACP synthesis. Sequencing of the acpS gene from the E. coli MP4 strain (denoted acpS1) showed that the AcpS1 protein contains a G4D mutation. AcpS1 exhibited a approximately 5-fold reduction in its catalytic efficiency when compared with wild type AcpS, accounting for the E. coli MP4 strain phenotype. It is shown that a conditional acpS mutant accumulates apoACP in vivo under nonpermissive conditions in a manner similar to the E. coli MP4 strain. In addition, it is demonstrated that the gene product, YhhU, of a previously identified E. coli open reading frame can completely suppress the acpS conditional, lethal phenotype upon overexpression of the protein, suggesting that YhhU may be involved in an alternative pathway for phosphopantetheinyl transfer and holoACP synthesis in E. coli.     

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from gamma-aminobutyrate and glutamate

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway.     

Analysis of in vivo substrate specificity of the PHA synthase from Ralstonia eutropha: formation of novel copolyesters in recombinant Escherichia coli

In order to investigate the in vivo substrate specificity of the type I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha, we functionally expressed the PHA synthase gene in various Escherichia coli mutants affected in fatty acid beta-oxidation and the wild-type. The PHA synthase gene was expressed either solely (pBHR70) or in addition to the R. eutropha genes encoding beta-ketothiolase and acetoacetyl-coenzyme A (CoA) reductase comprising the entire PHB operon (pBHR68) as well as in combination with the phaC1 gene (pBHR77) from Pseudomonas aeruginosa encoding type II PHA synthase. The fatty acid beta-oxidation route was employed to provide various 3-hydroxyacyl-CoA thioesters, depending on the carbon source, as in vivo substrate for the PHA synthase. In vivo PHA synthase activity was indicated by PHA accumulation and substrate specificity was revealed by analysis of the comonomer composition of the respective polyester. Only in recombinant E. coli fad mutants harboring plasmid pBHR68, the R. eutropha PHA synthase led to accumulation of poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) (poly(3HB-co-3HO)) and poly(3HB-co-3HO-co-3-hydroxydodecanoate (3HDD)), when octanoate and decanoate or dodecanoate were provided as carbon source, respectively. Coexpression of phaC1 from P. aeruginosa indicated and confirmed the provision of PHA precursor via the beta-oxidation pathway and led to the accumulation of a blend of two different PHAs in the respective E. coli strain. These data strongly suggested that R. eutropha PHA synthase accepts, besides the main substrate 3-hydroxybutyryl-CoA, also the CoA thioesters of 3HO and 3HDD.     

Regulation of coenzyme A biosynthesis.

Coenzyme A (CoA) and acyl carrier protein are two cofactors in fatty acid metabolism, and both possess a 4'-phosphopantetheine moiety that is metabolically derived from the vitamin pantothenate. We studied the regulation of the metabolic pathway that gives rise to these two cofactors in an Escherichia coli beta-alanine auxotroph, strain SJ16. Identification and quantitation of the intracellular and extracellular beta-alanine-derived metabolites from cells grown on increasing beta-alanine concentrations were performed. The intracellular content of acyl carrier protein was relatively insensitive to beta-alanine input, whereas the CoA content increased as a function of external beta-alanine concentration, reaching a maximum at 8 microM beta-alanine. Further increase in the beta-alanine concentration led to the excretion of pantothenate into the medium. Comparing the amount of pantothenate found outside the cell to the level of intracellular metabolites demonstrates that E. coli is capable of producing 15-fold more pantoic acid than is required to maintain the intracellular CoA content. Therefore, the supply of pantoic acid is not a limiting factor in CoA biosynthesis. Wild-type cells also excreted pantothenate into the medium, showing that the beta-alanine supply is also not rate limiting in CoA biogenesis. Taken together, the results point to pantothenate kinase as the primary enzymatic step that regulates the CoA content of E. coli.     

Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F

The gene encoding porin protein F of Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed as the predominant outer membrane protein in a porin-deficient E. coli background and was clearly visible on one-dimensional sodium dodecyl sulfate-polyacrylamide gels in a porin-sufficient background. The identity of the protein F from the E. coli clone and native P. aeruginosa protein F was demonstrated by their identical mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoretograms, 2-mercaptoethanol modifiabilities, and reactivities with monoclonal antibodies specific of two separate epitopes of protein F. In the course of gene subcloning, a 2-kilobase DNA fragment was isolated, with an apparent truncation of the part of the gene encoding the carboxy terminus of protein F. This subclone produced a 24,000-molecular-weight, outer membrane-associated, truncated protein F derivative which was not 2-mercaptoethanol modifiable and which reacted with only one of the two classes of protein F-specific monoclonal antibodies. The 2-kilobase fragment was used in Southern blot hybridizations to construct a restriction map of the cloned and subcloned fragments and to demonstrate with restriction digests of whole P. aeruginosa DNA that only one copy of the protein F gene was present in the P. aeruginosa chromosome. The protein F produced by the original cosmid clone in a porin-deficient E. coli background was purified. To demonstrate retention of porin function after cloning, the protein F from the E. coli clone was incorporated into black lipid bilayer membranes. Two major classes of channels were revealed. The predominant class of channels had an average conductance of 0.36 nS in 1 M KCl, whereas larger channels (4 to 7 nS) were seen at a lower frequency. Similar channel sizes were observed for porin protein F purified by the same method from P. aeruginosa outer membranes.     

FabG, an NADPH-dependent 3-ketoacyl reductase of Pseudomonas aeruginosa, provides precursors for medium-chain-length poly-3-hydroxyalkanoate biosynthesis in Escherichia coli

Escherichia coli hosts expressing fabG of Pseudomonas aeruginosa showed 3-ketoacyl coenzyme A (CoA) reductase activity toward R-3-hydroxyoctanoyl-CoA. Furthermore, E. coli recombinants carrying the poly-3-hydroxyalkanoate (PHA) polymerase-encoding gene phaC in addition to fabG accumulated medium-chain-length PHAs (mcl-PHAs) from alkanoates. When E. coli fadB or fadA mutants, which are deficient in steps downstream or upstream of the 3-ketoacyl-CoA formation step during beta-oxidation, respectively, were transformed with fabG, higher levels of PHA were synthesized in E. coli fadA, whereas similar levels of PHA were found in E. coli fadB, compared with those of the corresponding mutants carrying phaC alone. These results strongly suggest that FabG of P. aeruginosa is able to reduce mcl-3-ketoacyl-CoAs generated by the beta-oxidation to 3-hydroxyacyl-CoAs to provide precursors for the PHA polymerase.     

Metabolism of 4''-phosphopantetheine in Escherichia coli.

Coenzyme A (CoA) and acyl carrier protein (ACP) contain 4'-phosphopantetheine moieties that are metabolically derived from the vitamin pantothenate. The utilization of metabolites in the biosynthetic pathway during growth was investigated by using an Escherichia coli beta-alanine auxotroph to specifically and uniformly label the pathway intermediates. Pantothenate and 4'-phosphopantetheine were the two intermediates detected in the highest concentration, both intracellularly and extracellularly. The specific cellular content of CoA and ACP was not constant during growth of strain SJ16 (panD) on 4 microM beta-[3-3H]alanine, and alterations in the utilization of 4'-phosphopantetheine and pantothenate correlated with the observed fluctuations of the intracellular pool sizes of CoA and ACP. Double-label experiments indicated that extracellular 4'-phosphopantetheine was derived from the degradation of ACP, and the extent that this intermediate was utilized by 4'-phosphopantetheine adenylyltransferase exerted control over the degradative aspect of the pathway. Control over the biosynthetic aspect of the biochemical pathway was exerted at the level of pantothenate utilization by pantothenate kinase. Reduction in the specific cellular content of CoA and ACP by 4'-phosphopantetheine excretion was irreversible since, in contrast to pantothenate, strain SJ16 was unable to assimilate exogenous 4'-phosphopantetheine into CoA or ACP.     

An Arabidopsis mutant impaired in coenzyme A biosynthesis is sugar dependent for seedling establishment

Once the plant coenzyme A (CoA) biosynthetic pathway has been elucidated by comparative genomics, it is feasible to analyze the physiological relevance of CoA biosynthesis in plant life. To this end, we have identified and characterized Arabidopsis (Arabidopsis thaliana) T-DNA knockout mutants of two CoA biosynthetic genes, HAL3A and HAL3B. The HAL3A gene encodes a 4'-phosphopantothenoyl-cysteine decarboxilase that generates 4'-phosphopantetheine. A second gene, HAL3B, whose gene product is 86% identical to that of HAL3A, is present in the Arabidopsis genome. HAL3A appears to have a predominant role over HAL3B according to their respective mRNA expression levels. The hal3a-1, hal3a-2, and hal3b mutants were viable and showed a similar growth rate as that in wild-type plants; in contrast, a hal3a-1 hal3b double mutant was embryo lethal. Unexpectedly, seedlings that were null for HAL3A and heterozygous for HAL3B (aaBb genotype) displayed a sucrose (Suc)-dependent phenotype for seedling establishment, which is in common with mutants defective in beta-oxidation. This phenotype was genetically complemented in aaBB siblings of the progeny and chemically complemented by pantethine. In contrast, seedling establishment of Aabb plants was not Suc dependent, proving a predominant role of HAL3A over HAL3B at this stage. Total fatty acid and acyl-CoA measurements of 5-d-old aaBb seedlings in medium lacking Suc revealed stalled storage lipid catabolism and impaired CoA biosynthesis; in particular, acetyl-CoA levels were reduced by approximately 80%. Taken together, these results provide in vivo evidence for the function of HAL3A and HAL3B, and they point out the critical role of CoA biosynthesis during early postgerminative growth.     

Comparative studies of penicillin-binding proteins in Pseudomonas aeruginosa and Escherichia coli.

Penicillin-binding proteins in Pseudomonas aeruginosa were compared with those of Escherichia coli. These in P. aeruginosa were found exclusively in the cytoplasmic membrane fraction (fraction soluble in sodium N-lauroyl sarcosinate). Sodium dodecyl sulfate/acrylamide gel electrophoresis of the proteins bound to [14C]penicillin G resulted in the separation of six major bands and several minor bands. The proteins in these bands are referred to as proteins 1A, 1B, 2, 3, 4 and 5 in order of increasing electrophoretical mobility. The electrophoretic mobilities and other properties of penicillin-binding proteins in P. aeruginosa and E. coli were compared and correlated. Fundamentally they seem to be very similar in the two bacteria, but proteins 1A and 1B in P. aeruginosa seem to correspond respectively to proteins 1B and 1A in E. coli, and protein 6 seems to be missing or present in only small amount in P. aeruginosa. In addition, the affinities of currently developed beta-lactam antibiotics to each protein of P. aeruginosa and E. coli were examined in relation to the morphological changes of the cells induced by these antibiotics and their antibacterial potencies. Mecillinam showed high affinity to only protein 2 in both P. aeruginosa and E. coli. At a minimal inhibitory concentration, it converted cells of both P. aeruginosa and E. coli from rods to spherical cells, although its minimal inhibitory concentration was much higher for P. aeruginosa than for E. coli.     

Precursor-directed biosynthesis of stilbene methyl ethers in Escherichia coli

Stilbenes are bioactive compounds that show beneficial effects for humans, such as anti-tumor activity and survival improvement. Resveratrol, a representative of stilbenes and showing various health-improving activities, is rapidly metabolized in humans, and modified resveratrols are therefore desired as anti-cancer drugs and dietary polyphenols. An Escherichia coli system, in which an artificial stilbene biosynthetic pathway, including steps of phenylalanine ammonia-lyase, 4-coumarate:CoA ligase, and stilbene synthase, was reconstructed, produced stilbenes in high yields: resveratrol from tyrosine and pinosylvin from phenylalanine. To incorporate a stilbene methyltransferase gene into this E. coli system, cDNA of Os08g06100 in Oryza sativa was expressed and its O-methylating activity toward stilbenes was confirmed. Incorporation of the pinosylvin methyltransferase (OsPMT) gene into the pathway established in E. coli led to production of mono- and di-methylated stilbenes. Furthermore, the OsPMT gene turned out to be useful in production of unnatural stilbene methyl ethers due to its rather relaxed substrate specificity; various carboxylic acids supplemented as precursors, such as p-fluorocinnamic acid, 3-(2-furyl)acrylic acid, 3-(2-thienyl)acrylic acid, and 3-(3-pyridyl)acrylic acid, to the E. coli system carrying the steps of 4-coumarate:CoA ligase, stilbene synthase, and OsPMT were converted to stilbene dimethyl ethers with the corresponding carboxylic moiety.     

Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa.

A mutant of Pseudomonas aeruginosa was characterized which could not grow anaerobically with nitrate as the terminal electron acceptor or with arginine as the sole energy source. In this anr mutant, nitrate reductase and arginine deiminase were not induced by oxygen limitation. The anr mutation was mapped in the 60-min region of the P. aeruginosa chromosome. A 1.3-kb chromosomal fragment from P. aeruginosa complemented the anr mutation and also restored anaerobic growth of an Escherichia coli fnr deletion mutant on nitrate medium, indicating that the 1.3-kb fragment specifies an FNR-like regulatory protein. The arcDABC operon, which encodes the arginine deiminase pathway enzymes of P. aeruginosa, was rendered virtually noninducible by a deletion or an insertion in the -40 region of the arc promoter. This -40 sequence (TTGAC....ATCAG) strongly resembled the consensus FNR-binding site (TTGAT....ATCAA) of E. coli. The cloned arc operon was expressed at low levels in E. coli; nevertheless, some FNR-dependent anaerobic induction could be observed. An FNR-dependent E. coli promoter containing the consensus FNR-binding site was expressed well in P. aeruginosa and was regulated by oxygen limitation. These findings suggest that P. aeruginosa and E. coli have similar mechanisms of anaerobic control.     

Crystal structure of a type III pantothenate kinase: insight into the mechanism of an essential coenzyme A biosynthetic enzyme universally distributed in bacteria

Pantothenate kinase (PanK) catalyzes the first step in the five-step universal pathway of coenzyme A (CoA) biosynthesis, a key transformation that generally also regulates the intracellular concentration of CoA through feedback inhibition. A novel PanK protein encoded by the gene coaX was recently identified that is distinct from the previously characterized type I PanK (exemplified by the Escherichia coli coaA-encoded PanK protein) and type II eukaryotic PanKs and is not inhibited by CoA or its thioesters. This type III PanK, or PanK-III, is widely distributed in the bacterial kingdom and accounts for the only known PanK in many pathogenic species, such as Helicobacter pylori, Bordetella pertussis, and Pseudomonas aeruginosa. Here we report the first crystal structure of a type III PanK, the enzyme from Thermotoga maritima (PanK(Tm)), solved at 2.0-A resolution. The structure of PanK(Tm) reveals that type III PanKs belong to the acetate and sugar kinase/heat shock protein 70/actin (ASKHA) protein superfamily and that they retain the highly conserved active site motifs common to all members of this superfamily. Comparative structural analysis of the PanK(Tm) active site configuration and mutagenesis of three highly conserved active site aspartates identify these residues as critical for PanK-III catalysis. Furthermore, the analysis also provides an explanation for the lack of CoA feedback inhibition by the enzyme. Since PanK-III adopts a different structural fold from that of the E. coli PanK -- which is a member of the "P-loop kinase"superfamily -- this finding represents yet another example of convergent evolution of the same biological function from a different protein ancestor.     

Applicability of CoA/acetyl-CoA manipulation system to enhance isoamyl acetate production in Escherichia coli

Coenzyme A (CoA) and its thioester derivatives are important precursor molecules for many industrially useful compounds such as esters, PHBs, lycopene and polyketides. Previously, in our lab we could increase the intracellular levels of CoA and acetyl-Coenzyme A (acetyl-CoA) by overexpressing one of the upstream rate-controlling enzymes pantothenate kinase with a concomitant supplementation of the precursor pantothenic acid to the cell culture medium. In this study, we showed that the CoA/acetyl-CoA manipulation system could be used to increase the productivity of industrially useful compounds derived from acetyl-CoA. We chose the production of isoamyl acetate as a model system. Isoamyl acetate is an important flavor component of sake yeast and holds a great commercial value. Alcohol acetyl transferase (AAT) condenses isoamyl alcohol and acetyl-CoA to produce isoamyl acetate. The gene ATF2, coding for this AAT was cloned and expressed in Escherichia coli. This genetic engineered E. coli produces isoamyl acetate, an ester, from intracellular acetyl-CoA when isoamyl alcohol is added externally to the cell culture medium. In the current study, we showed that in a strain bearing ATF2 gene, an increase in intracellular CoA/acetyl-CoA by overexpressing panK leads to an increase in isoamyl acetate production. Additionally, the cofactor manipulation technique was combined with more traditional approach of competing pathway deletions to further increase isoamyl acetate production. The acetate production pathway competes with isoamyl acetate production for the common intracellular metabolite acetyl-CoA. Earlier we have shown that acetate pathway deletion (ackA-pta) increases isoamyl acetate production. The acetate production pathway was inactivated under elevated CoA/acetyl-CoA conditions, which lead to a further increase in isoamyl acetate production.     

Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus

Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated beta-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and (1)H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of beta-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.     

The antibiotic activity of N-pentylpantothenamide results from its conversion to ethyldethia-coenzyme a,a coenzyme a antimetabolite

Pantothenic acid (vitamin B(5)) is the natural precursor of coenzyme A (CoA), an essential cofactor in all organisms. The pantothenic acid antimetabolite N-pentylpantothenamide inhibits the growth of Escherichia coli with a minimum inhibitory concentration of 2 microm. In this study, we examine the mechanism of this inhibition. Using the last five enzymes of the CoA biosynthetic pathway in E. coli we demonstrate that N-pentylpantothenamide does not inhibit the CoA biosynthetic enzymes but instead acts as an alternative substrate, forming the CoA analog ethyldethia-CoA. We show that N-pentylpantothenamide is converted to ethyldethia-CoA 10.5 times faster than CoA is biosynthesized from pantothenic acid, demonstrating that ethyldethia-CoA biosynthesis can effectively compete with CoA biosynthesis in the cell. We conclude that the mechanism of toxicity of N-pentylpantothenamide is most likely due to its biosynthetic conversion to the CoA analog ethyldethia-CoA, which may act as an inhibitor of CoA- and acetyl-CoA-utilizing enzymes.     

Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium

The human bile acid pool composition is composed of both primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acids (deoxycholic acid and lithocholic acid). Secondary bile acids are formed by the 7α-dehydroxylation of primary bile acids carried out by intestinal anaerobic bacteria. We have previously described a multistep biochemical pathway in Clostridium scindens that is responsible for bile acid 7α-dehydroxylation. We have identified a large (12 kb) bile acid inducible (bai) operon in this bacterium that encodes eight genes involved in bile acid 7α-dehydroxylation. However, the function of the baiF gene product in this operon has not been elucidated. In the current study, we cloned and expressed the baiF gene in E. coli and discovered it has bile acid CoA transferase activity. In addition, we discovered a second bai operon encoding three genes. The baiK gene in this operon was expressed in E. coli and found to encode a second bile acid CoA transferase. Both bile acid CoA transferases were determined to be members of the type III family by amino acid sequence comparisons. Both bile acid CoA transferases had broad substrate specificity, except the baiK gene product, which failed to use lithocholyl-CoA as a CoA donor. Primary bile acids are ligated to CoA via an ATP-dependent mechanism during the initial steps of 7α-dehydroxylation. The bile acid CoA transferases conserve the thioester bond energy, saving the cell ATP molecules during bile acid 7α-dehydroxylation. ATP-dependent CoA ligation is likely quickly supplanted by ATP-independent CoA transfer.     

Comparative action of glyphosate as a trigger of energy drain in eubacteria.

Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, each possessing a 5-enolpyruvylshikimate 3-phosphate synthase that is sensitive to inhibition by glyphosate [N-(phosphonomethyl)glycine], provide a good cross-section of organisms exemplifying the biochemical diversity of the aromatic pathway targeted by this potent antimicrobial compound. The pattern of growth inhibition, the alteration in levels of aromatic-pathway enzymes, and the accumulation of early-pathway metabolites after the addition of glyphosate were distinctive for each organism. Substantial intracellular shikimate-3-phosphate accumulated in response to glyphosate treatment in all three organisms. Both E. coli and P. aeruginosa, but not B. subtilis, accumulated near-millimolar levels of shikimate-3-phosphate in the culture medium. Intracellular backup of common-pathway precursors of shikimate-3-phosphate was substantial in B. subtilis, moderate in P. aeruginosa, and not detectable in E. coli. The full complement of aromatic amino acids prevented growth inhibition and metabolite accumulation in E. coli and P. aeruginosa where amino acid end products directly control early-pathway enzyme activity. In contrast, the initial prevention of growth inhibition in the presence of aromatic amino acids in B. subtilis was succeeded by progressively greater growth inhibition that correlated with rapid metabolite accumulation. In B. subtilis glyphosate can decrease prephenate concentrations sufficiently to uncouple the sequentially acting loops of feedback inhibition that ordinarily link end product excess to feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by prephenate. The consequential unrestrained entry is an energy-rich substrates into the aromatic pathway, even in the presence of aromatic amino acid end products, is an energy drain that potentially accounts for the inability of end products to fully reverse glyphosate inhibition in B. subtilis. Even in E. coli after glyphosate inhibition and metabolite accumulation were allowed to become fully established, a transient period where end products were capable of only partial reversal of growth inhibition occurred. The distinctive metabolism produced by dissimilation of different carbon sources also profound effects upon glyphosate sensitivity.     

Biochemical characterization of a cinnamoyl-CoA reductase from wheat

Cinnamoyl-CoA reductase (CCR) is responsible for the CoA ester-->aldehyde conversion in monolignol biosynthesis, which can divert phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of lignin biosynthesis in wheat (Triticum aestivum L.), a cDNA encoding CCR was isolated and named Ta-CCR2. DNA hybridization analyses demonstrated that the Ta-CCR2 gene exists in three copies in the wheat genome. RNA blot hybridization indicated that Ta-CCR2 was expressed most abundantly in root and stem tissues that were in the process of lignification. The secondary and three-dimensional structures of Ta-CCR2 were analyzed by molecular modeling. Recombinant Ta-CCR2 protein purified from E. coli converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA and caffeoyl CoA with almost similar efficiency, suggesting that it is involved in both G and S lignin synthesis. Ta-CCR2 had a very low V max value for 4-coumaroyl CoA, which may serve as a mechanism to control metabolic flux to H lignin in vivo . Furthermore, the reaction mechanism of Ta-CCR2 was analyzed in relation to its possible three-dimensional structure. The activity of Ta-CCR2 in relation to lignin biosynthesis is discussed.