Dietary Catechols and their Relationship to Microbial Endocrinology.

This chapter examines the evidence that the ability of neuroendocrine hormones, notably norepinephrine and epinephrine, to stimulate bacterial growth in iron-restricted media is not limited to molecules with a catecholamine structure but is also possessed by a variety of other catechols, many of which are of plant origin and are common in the diet. Catechols derived from the diet, such as the tea flavanols, can be present in the plasma at submicromolar and micromolar concentrations, comparable with the concentrations of catecholamines that have been shown to be effective in promoting bacterial growth under conditions of iron restriction, although many dietary catechols, notably quercetin derivatives, are present in the plasma and tissues largely as conjugates, from which the catechol function has been lost. Finally, although bacterial growth promotion through relief of iron restriction appears to be exhibited by a wide range of catechols, the gene-activation effects of catecholamines demonstrated to occur in some bacteria may be much more specific, although the definitive experiments to establish structure-function relationships have yet to be reported.

Network analysis of the transcriptional pattern of young and old cells of Escherichia coli during lag phase.

BACKGROUND: The aging process of bacteria in stationary phase is halted if cells are subcultured and enter lag phase and it is then followed by cellular division. Network science has been applied to analyse the transcriptional response, during lag phase, of bacterial cells starved previously in stationary phase for 1 day (young cells) and 16 days (old cells). RESULTS: A genome scale network was constructed for E. coli K-12 by connecting genes with operons, transcription and sigma factors, metabolic pathways and cell functional categories. Most of the transcriptional changes were detected immediately upon entering lag phase and were maintained throughout this period. The lag period was longer for older cells and the analysis of the transcriptome revealed different intracellular activity in young and old cells. The number of genes differentially expressed was smaller in old cells (186) than in young cells (467). Relatively, few genes (62) were up- or down-regulated in both cultures. Transcription of genes related to osmotolerance, acid resistance, oxidative stress and adaptation to other stresses was down-regulated in both young and old cells. Regarding carbohydrate metabolism, genes related to the citrate cycle were up-regulated in young cells while old cells up-regulated the Entner Doudoroff and gluconate pathways and down-regulated the pentose phosphate pathway. In both old and young cells, anaerobic respiration and fermentation pathways were down-regulated, but only young cells up-regulated aerobic respiration while there was no evidence of aerobic respiration in old cells.Numerous genes related to DNA maintenance and replication, translation, ribosomal biosynthesis and RNA processing as well as biosynthesis of the cell envelope and flagellum and several components of the chemotaxis signal transduction complex were up-regulated only in young cells. The genes for several transport proteins for iron compounds were up-regulated in both young and old cells. Numerous genes encoding transporters for carbohydrates and organic alcohols and acids were down-regulated in old cells only. CONCLUSION: Network analysis revealed very different transcriptional activities during the lag period in old and young cells. Rejuvenation seems to take place during exponential growth by replicative dilution of old cellular components.

A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism.

HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and is exploited in whole-cell systems for the bioconversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K(M) for the substrate ferulic acid, fulfilling its anticipated role as a factor in substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.

Riboflavin biosynthesis is associated with assimilatory ferric reduction and iron acquisition by Campylobacter jejuni.

One of the pathways involved in the acquisition of the essential metal iron by bacteria involves the reduction of insoluble Fe(3+) to soluble Fe(2+), followed by transport of Fe(2+) to the cytoplasm. Flavins have been implicated as electron donors in this poorly understood process. Ferrous iron uptake is essential for intestinal colonization by the important pathogen Campylobacter jejuni and may be of particular importance under low-oxygen conditions. In this study, the links among riboflavin biosynthesis, ferric reduction, and iron acquisition in C. jejuni NCTC11168 have been investigated. A riboflavin auxotroph was generated by inactivation of the ribB riboflavin biosynthesis gene (Cj0572), and the resulting isogenic ribB mutant only grew in the presence of exogenous riboflavin or the riboflavin precursor diacetyl but not in the presence of the downstream products flavin adenine dinucleotide and flavin mononucleotide. Riboflavin uptake was unaffected in the ribB mutant under iron-limited conditions but was lower in both the wild-type strain and the ribB mutant under iron-replete conditions. Mutation of the fur gene, which encodes an iron uptake regulator of C. jejuni, resulted in an increase in riboflavin uptake which was independent of the iron content of the medium, suggesting a role for Fur in the regulation of the as-yet-unknown riboflavin transport system. Finally, ferric reduction activity was independent of iron availability in the growth medium but was lowered in the ribB mutant compared to the wild-type strain and, conversely, increased in the fur mutant. Taken together, the findings confirm close relationships among iron acquisition, riboflavin production, and riboflavin uptake in C. jejuni.

Influence of dietary catechols on the growth of enteropathogenic bacteria.

The dietary constituents that may act, in the broadest sense, as co-factors to enable bacterial enteropathogens to replicate in gastrointestinal environments are still largely unknown. Recent work has demonstrated that certain non-nutritional components of food, such as the catecholamines, can contribute to the ability of Gram-negative pathogens to replicate in iron-restrictive media that may be reflective of gastrointestinal environments. The present report examines whether other, non-catecholamine, dietary catechols, which occur widely in plant foods, can also influence enteropathogen growth in an iron-restrictive environment such as might be found in the gastrointestinal tract. In the present study, we have examined the ability of a range of catechol-rich foodstuffs, ranging from beverages (tea and coffee) to fruit and vegetable extracts, as well as purified preparations of commonly consumed dietary catechols (catechins, chlorogenic acid, caffeic acid and tannic acid), to modulate the growth of the Gram-negative enteric pathogens Escherichia coli O157:H7 and Salmonella enterica SV Enteriditis. Time-dependent growth in response to dietary catechols (0.05-5.0% v/v of beverage or fruit/vegetable extracts; 10-200 microM of purified catechols) was examined in an iron-replete, rich medium as well as in an iron-limited, basal medium designed to reflect the iron-restricted environment that is more characteristic of human and animal tissues. Results obtained in iron-replete, rich medium demonstrated dose-dependent bacteriostatic effects for certain catechols, consistent with previous studies. However, in iron-restricted medium, all of the dietary catechols produced marked growth stimulation of up to 4 logs greater than non-supplemented controls. Mechanistic studies measuring the uptake of radiolabelled (55)Fe from (55)Fe-labelled lactoferrin and transferrin in bacteria grown in the presence or absence of dietary catechols demonstrated that the ability of catechols to stimulate bacterial growth was dependent on the provision of iron from iron-sequestering glycoproteins. Urea gel analysis of transferrin incubated in the presence of the dietary catechols confirmed that these compounds were directly chelating and removing transferrin-complexed iron. Analysis using E. coli O157:H7 entA and tonB mutants further showed that a functional siderophore synthesis and uptake system was required for the growth-stimulatory response. In contrast to previous studies, which have reported the anti-microbial activity of dietary catechols, the present study demonstrates that these non-nutritional components of foods can, under iron-restrictive conditions, provide iron and enable the growth of enteric bacterial pathogens.

The 1.8 A resolution structure of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL) from Pseudomonas fluorescens, an enzyme that catalyses the transformation of feruloyl-coenzyme A to vanillin.

The crystal structure of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) from Pseudomonas fluorescens AN103 has been solved to 1.8 A resolution. HCHL is a member of the crotonase superfamily and catalyses the hydration of the acyl-CoA thioester of ferulic acid [3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid] and the subsequent retro-aldol cleavage of the hydrated intermediate to yield vanillin (4-hydroxy-3-methoxy-benzaldehyde). The structure contains 12 molecules in the asymmetric unit, in which HCHL assumes a hexameric structure of two stacked trimers. The substrate, feruloyl-CoA, was modelled into the active site based on the structure of enoyl-CoA hydratase bound to the feruloyl-CoA-like substrate 4-(N,N-dimethylamino)-cinnamoyl-CoA (PDB code 1ey3). Feruloyl-CoA was bound in this model between helix 3 of the A subunit and helix 9 of the B subunit. A highly ordered structural water in the HCHL structure coincided with the thioester carbonyl of feruloyl-CoA in the model, suggesting that the oxyanion hole for stabilization of a thioester-derived enolate, characteristic of coenzyme-A dependent members of the crotonase superfamily, is conserved. The model also suggested that a strong hydrogen bond between the phenolic hydroxyl groups of feruloyl-CoA and BTyr239 may be an important determinant of the enzyme's ability to discriminate between the natural substrate and cinnamoyl-CoA, which is not a substrate.

Initial evaluation of sugarcane as a production platform for p-hydroxybenzoic acid.

Sugarcane (Saccharum hybrids) was evaluated as a production platform for p-hydroxybenzoic acid using two different bacterial proteins (a chloroplast-targeted version of Escherichia coli chorismate pyruvate-lyase and 4-hydroxycinnamoyl-CoA hydratase/lyase from Pseudomonas fluorescens) that both provide a one-enzyme pathway from a naturally occurring plant intermediate. The substrates for these enzymes are chorismate (a shikimate pathway intermediate that is synthesized in plastids) and 4-hydroxycinnamoyl-CoA (a cytosolic phenylpropanoid intermediate). Although both proteins have previously been shown to elevate p-hydroxybenzoic acid levels in plants, they have never been evaluated concurrently in the same laboratory. Nor are there any reports on their efficacy in stem tissue. After surveying two large populations of transgenic plants, it was concluded that the hydratase/lyase is the superior catalyst for leaf and stem tissue, and further studies focused on this pathway. p-Hydroxybenzoic acid was quantitatively converted to glucose conjugates by endogenous uridine diphosphate (UDP)-glucosyltransferases and presumably stored in the vacuole. The largest amounts detected in leaf and stem tissue were 7.3% and 1.5% dry weight (DW), respectively, yet there were no discernible phenotypic abnormalities. However, as a result of diverting carbon away from the phenylpropanoid pathway, there was a severe reduction in leaf chlorogenic acid, subtle changes in lignin composition, as revealed by phloroglucinol staining, and an apparent compensatory up-regulation of phenylalanine ammonia-lyase. Although product accumulation in the leaves at the highest level of gene expression obtained in the present study was clearly substrate-limited, additional experiments are necessary before this conclusion can be extended to the stalk.

Purification, crystallization and preliminary X-ray crystallographic analysis of hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), a crotonase homologue active in phenylpropanoid metabolism.

4-Hydroxycinnamoyl-coenzyme A hydratase-lyase (HCHL), also called feruloyl-CoA hydratase-lyase (FCHL), from Pseudomonas fluorescens strain AN103 is an enzyme of the crotonase superfamily that catalyses the one-step conversion of the CoA thioesters of 4-coumaric acid, caffeic acid and ferulic acid to the aromatic aldehydes 4-hydroxybenzaldehyde, protocatechuic aldehyde and vanillin, respectively. The reaction occurs via a hydration followed by a carbon-carbon bond-cleavage reaction. HCHL has been crystallized by the hanging-drop method of vapour diffusion using polyethylene glycol 20 000 Da as the precipitant. The crystals belong to the orthorhombic system, with proposed space group P2(1)2(1)2 and unit-cell parameters a = 154.2, b = 167.5, c = 130.8 A. The V(M) suggests that the asymmetric unit contains four trimers. Single-wavelength data collection has been undertaken and structure determination is under way by molecular replacement using data collected to 1.8 A resolution. Determination of the structure of HCHL will provide insight into the catalytic mechanism of an unusual enzymatic reaction with relevance to the applications of the enzyme in metabolic engineering.

The diverse bacterial origins of the Arabidopsis polyamine biosynthetic pathway.

We functionally identified the last remaining step in the plant polyamine biosynthetic pathway by expressing an Arabidopsis thaliana agmatine iminohydrolase cDNA in yeast. Inspection of the whole pathway suggests that the arginine decarboxylase, agmatine iminohydrolase, N-carbamoylputrescine amidohydrolase route to putrescine in plants was inherited from the cyanobacterial ancestor of the chloroplast. However, the rest of the pathway including ornithine decarboxylase and spermidine synthase was probably inherited from bacterial genes present in the original host cell, common ancestor of plants and animals, that acquired the cyanobacterial endosymbiont. An exception is S-adenosylmethionine decarboxylase, which may represent a eukaryote-specific enzyme form.

Vanillin.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an important flavour and aroma molecule, but is also of interest because of its biogenetic relationship to the phenylpropanoid pathway and to other molecules of physiological significance, notably salicylate. Recent progress towards characterisation of the biosynthesis of vanillin is reviewed. In Vanilla, there is some evidence that the route to vanillin-beta-D-glucoside may proceed from 4-coumaric acid via 4-hydroxybenzaldehyde, with glucoside formation occurring not necessarily as the final step, and possibly with the involvement of 4-hydroxybenzyl alcohol beta-D-glucoside tartrate bis-esters as "shunt" metabolites. This appears to be given tentative support by the recent partial characterisation of a 4-hydroxybenzaldehyde synthase from Vanilla. On the other hand, a well-characterised, CoA-dependent, non-oxidative chain-shortening mechanism to produce vanillin from ferulic acid, occurring as part of a pathway of hydroxycinnamate degradation in Pseudomonas, may not be representative of hydroxycinnamate chain-shortening mechanism(s) occurring in Vanilla and other plants. Nevertheless, by expression of the Pseudomonas enzyme 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), attempts have been made to introduce a direct capacity for vanillin formation into model plants by diversion of the phenylpropanoid pathway. The results obtained have emphasised the obstacles to achieving the desired oxidation level (aldehyde) and ring substitution (4-hydroxy-3-methoxyphenyl), even when a substantial metabolic diversion is successfully achieved. Finally, the significance of the latest biosynthetic and biotechnological developments is reviewed briefly in relation to authentication of vanillin.

4-Hydroxycinnamoyl-CoA hydratase/lyase, an enzyme of phenylpropanoid cleavage from Pseudomonas, causes formation of C(6)-C(1) acid and alcohol glucose conjugates when expressed in hairy roots of Datura stramonium L.

4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL), a crotonase homologue of phenylpropanoid catabolism from Pseudomonas fluorescens strain AN103, led to the formation of 4-hydroxybenzaldehyde metabolites when expressed in hairy root cultures of Datura stramonium L. established by transformation with Agrobacterium rhizogenes. The principal new compounds observed were the glucoside and glucose ester of 4-hydroxybenzoic acid, together with 4-hydroxybenzyl alcohol- O-beta- D-glucoside. In lines actively expressing HCHL, these together amounted to around 0.5% of tissue fresh mass. No protocatechuic derivatives were found, although a trace of vanillic acid-beta- D-glucoside was detected. There was no accumulation of 4-hydroxybenzaldehydes, whether free or in the form of their glucose conjugates. There was some evidence suggesting a diminished availability of feruloyl-CoA for the production of feruloyl putrescine and coniferyl alcohol. The findings are discussed in the context of a diversion of phenylpropanoid metabolism, and the ability of plants and plant cultures to conjugate phenolic compounds.