Enzymes of secondary metabolism and the biosynthesis of macrolide antibiotics

Enzymes participating in the biosynthesis of macrolide antibiotics are reviewed. Enzyme activities are known to play a pivotal role in the formation of biologically active compounds. Hence it is essential to understand these enzymes, their properties and regulation. Macrolide antibiotics represent a relatively compact group of natural products and include several excellent model compounds suitable for enzyme studies that could be generalized to other oligoketide antibiotics.     

Catabolite repression control of pyocyanin biosynthesis at an intersection of primary and secondary metabolism in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the catabolite repression control (Crc) protein repressed the formation of the blue pigment pyocyanin in response to a preferred carbon source (succinate) by interacting with phzM mRNA, which encodes a key enzyme in pyocyanin biosynthesis. Crc bound to an extended imperfect recognition sequence that was interrupted by the AUG translation initiation codon.     

Molecular and metabolic control of secondary metabolism

Secondary metabolism is restricted to specific places in the plant. The concentrations of precursors and end products are the determining factors in the metabolic control of synthesis and breakdown of the compounds involved. Molecular control operates at the level of enzyme amount and gene expression. If the secondary product contains an element in its molecule which is derived from a mineral nutrient in the environment, the operation of the control mechanisms can be studied by varying the concentration of that mineral. This is exemplified by thiophene metabolism in root cultures ofTagetes. The characteristic groups in the molecule are two five-membered rings with a sulphur atom. In the experiments, the rate of thiophene biosynthesis was manipulated by varying the sulphate concentration in the medium. Sulphur limitation led to preferential channeling of sulphur into primary metabolism and a concomitant drop in thiophene biosynthesis. The major part of the reduction was caused by a drop in enzyme activity. Substrate availability played a minor role. The results indicate that sulphur is involved in the molecular control of secondary metabolism inTagetes.Abbreviations BBT 5-(but-3-en-l-ynyl)-2,2-bithienyl - BPT 2-(but-3-en-l-ynyl)-5-(penta-1,3-diynyl)-thiophene - PYE trideca-3,5,7,9,11-pentaynene     

TNF regulates cellular NAD+ metabolism in primary macrophages

The inflammatory cytokine TNF is known to affect glucose and lipid metabolism, where its action leads to a cachexic state. Despite a well-established connection of TNF to metabolism, the relationship between TNF and NAD(+) metabolism remains unclear. In this report, we evaluated the effects of TNF on NAD(+) metabolism in cells that are TNF's primary autocrine target-macrophages. We designed real-time PCR primers to all NAD(+) metabolic enzymes, which we used to examine TNF-induced changes over time. We found that TNF paradoxically up-regulated enzymes that served to increase NAD(+) levels, such as IDO and PBEF, as well as enzymes that decrease NAD(+) levels, such as CD38 and CD157. The significance of these mRNA changes was evaluated by examining TNF-mediated changes in cellular NAD(+) levels. Treatment of macrophages with TNF decreased NAD(+) levels over time, suggesting that increases in NAD(+)-degrading enzymes were dominant. To evaluate whether this was the case, we measured TNF-mediated changes in NAD(+) levels in animals where CD38 was genetically deleted. In CD38-/- macrophages, the effects of TNF were reversed, with TNF increasing NAD(+) levels over time. The significance of our findings is threefold: (1) we establish that TNF affects NAD(+) metabolism by regulating the expression of major NAD(+) metabolic enzymes, (2) TNF-induced decreases in cellular NAD(+) levels were carried out through the up-regulation of extracellularly situated enzymes, and (3) we provide a mechanism for the observed clinical connection of TNF-dependent diseases to tissue reductions in NAD(+) content.     

Primary and secondary metabolism regulates lipolysis in appressoria of Colletotrichum orbiculare

The conidia of Colletotrichum orbiculare, the causal agent of cucumber anthracnose, develop appressoria that are pigmented with melanin for host plant infection. Premature appressoria contain abundant lipid droplets (LDs), but these disappear during appressorial maturation, indicating lipolysis inside the appressorial cells. The lipolysis and melanization in appressoria require the peroxin PEX6, suggesting the importance of peroxisomal metabolism in these processes. To investigate the relationships between appressorial lipolysis and fungal metabolic pathways, C. orbiculare knockout mutants of MFE1, which encodes a peroxisomal multifunctional enzyme, were generated in this study, and the phenotype of the mfe1 mutants was investigated. In contrast to the wild-type strain, which forms melanized appressoria, the mfe1 mutants formed colorless nonmelanized appressoria with abundant LDs, similar to those of pex6 mutants. This indicates that fatty acid β-oxidation in peroxisomes is critical for the appressorial melanization and lipolysis of C. orbiculare. Soraphen A, a specific inhibitor of acetyl-CoA carboxylase, inhibited appressorial lipolysis and melanization, producing phenocopies of the mfe1 mutants. This suggests that the conversion of acetyl-CoA, derived from fatty acid β-oxidation, to malonyl-CoA is required for the activation of lipolysis in appressoria. Surprisingly, we found that genetically blocking PKS1-dependent polyketide synthesis, an initial step in melanin biosynthesis, also impaired appressorial lipolysis. In contrast, genetically or pharmacologically blocking the steps in melanin synthesis downstream from PKS1 did not abolish appressorial lipolysis. These findings indicate that melanin biosynthesis, as well as fatty acid β-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.     

Cyclic AMP regulation of tylosin biosynthesis and secondary metabolism in Streptomyces fradiae

Adenosine 3':5' cyclic monophosphate seems to regulate antibiotic biosynthesis and secondary metabolism in tylosin-producing cultures of Streptomyces fradiae C373.1. A dose-dependent response is observed by exogenous additions of dibutyryl cyclic AMP (cAMP), and is related to the nutritional status of the culture. Addition of cAMP to cultures growing in nutritionally lean media caused higher cumulative antibiotic tigers and some cellular differentiation compared with the control. In nutritionally rich media, a qualitatively different behavior resulted: an almost instantaneous shift toward secondary metabolism occurred. The response is characterized by extensive cellular differentiation with little growth and only a trace of antibiotic production. The possible role of cyclic AMP n the regulation of tylosin biosynthesis and secondary metabolism and its relation to specific nutrient limitations in synthetic, defined media in Streptomyces fradiae is discussed. (c) 1994 John Wiley & Sons, Inc.     

SUPERMAN regulates floral whorl boundaries through control of auxin biosynthesis

Proper floral patterning, including the number and position of floral organs in most plant species, is tightly controlled by the precise regulation of the persistence and size of floral meristems (FMs). In Arabidopsis, two known feedback pathways, one composed of WUSCHEL (WUS) and CLAVATA3 (CLV3) and the other composed of AGAMOUS (AG) and WUS, spatially and temporally control floral stem cells, respectively. However, mounting evidence suggests that other factors, including phytohormones, are also involved in floral meristem regulation. Here, we show that the boundary gene SUPERMAN (SUP) bridges floral organogenesis and floral meristem determinacy in another pathway that involves auxin signaling. SUP interacts with components of polycomb repressive complex 2 (PRC2) and fine‐tunes local auxin signaling by negatively regulating the expression of the auxin biosynthesis genes YUCCA1/4 (YUC1/4). In sup mutants, derepressed local YUC1/4 activity elevates auxin levels at the boundary between whorls 3 and 4, which leads to an increase in the number and the prolonged maintenance of floral stem cells, and consequently an increase in the number of reproductive organs. Our work presents a new floral meristem regulatory mechanism, in which SUP, a boundary gene, coordinates floral organogenesis and floral meristem size through fine‐tuning auxin biosynthesis.     

Modulation of cellular metabolism by protein crotonylation regulates pancreatic cancer progression

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Phosphinothricin-tripeptide biosynthesis: An original version of bacterial secondary metabolism?

Streptomyces viridochromogenes Tü494 produces the herbicide phosphinothricyl-alanyl-alanine (phosphinothricin-tripeptide = PTT; bialaphos). Its bioactive moiety phosphinothricin competitively inhibits bacterial and plant glutamine synthetases. The biosynthesis of PTT includes the synthesis of the unusual amino acid N-acetyl-demethyl-phosphinothricin and a three step condensation via non-ribosomal peptide synthetases. Two characteristics within the PTT biosynthesis make it suitable to study the evolution of secondary metabolism biosynthesis. First, PTT biosynthesis represents the only known system where all peptide synthetase modules are located on separate proteins. This ‘single enzyme system’ might be an archetype of the multimodular and multienzymatic non-ribosomal peptide synthetases in evolutionary terms. The second interesting feature of PTT biosynthesis is the pathway-specific aconitase Pmi that is involved in the supply of N-acetyl-demethyl-phosphinothricin. Pmi is highly similar to the tricarboxylic acid aconitase AcnA. They share 64% identity at the DNA level and both belong to the Iron-Regulatory-Protein/AcnA family. Despite their high sequence similarity, AcnA and Pmi catalyze different reactions and are not able to substitute for each other. Thus, the enzyme pair AcnA/Pmi presents an example of the evolution of a secondary metabolite-specific enzyme from a primary metabolism enzyme.     

Gene duplication in the diversification of secondary metabolism: tandem 2-oxoglutarate-dependent dioxygenases control glucosinolate biosynthesis in Arabidopsis

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions. The large chemical diversity of secondary metabolites undoubtedly arises from an equally diverse set of enzymes responsible for their biosynthesis. However, little is known about the evolution of enzymes involved in secondary metabolism. We are studying the biosynthesis of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate the evolution of enzymes involved in secondary metabolism. Arabidopsis contains natural variations in the presence of methylsulfinylalkyl, alkenyl, and hydroxyalkyl glucosinolates. In this article, we report the identification of genes encoding two 2-oxoglutarate--dependent dioxygenases that are responsible for this variation. These genes, AOP2 and AOP3, which map to the same position on chromosome IV, result from an apparent gene duplication and control the conversion of methylsulfinylalkyl glucosinolate to either the alkenyl or the hydroxyalkyl form. By heterologous expression in Escherichia and the correlation of gene expression patterns to the glucosinolate phenotype, we show that AOP2 catalyzes the conversion of methylsulfinylalkyl glucosinolates to alkenyl glucosinolates. Conversely, AOP3 directs the formation of hydroxyalkyl glucosinolates from methylsulfinylalkyl glucosinolates. No ecotype coexpressed both genes. Furthermore, the absence of functional AOP2 and AOP3 leads to the accumulation of the precursor methylsulfinylalkyl glucosinolates. A third member of this gene family, AOP1, is present in at least two forms and found in all ecotypes examined. However, its catalytic role is still uncertain.     

Glycinebetaine biosynthesis and its control in detached secondary leaves of spinach

In secondary leaves from spinach plants pretreated in vermiculite for 24 h with 300 mM NaCl, glycinebetaine accumulated at a rate of circa 0.16 mol 100 g^-1 Chl d^-1 (2 mol g^-1 FW d^-1), about three times the rate of control plants. The soluble carbohydrate and free amino acid contents did not increase significantly following salinisation until after 4 d when the relative growth rate also decreased. Leaf proline levels remained very low throughout the experimental period. K⁺ on a tissue water basis remained constant at 200 mM while Cl^- and Na⁺ levels increased linearly to reach 175 and 100 mM respectively after 5 d of saline treatment. The osmotic pressure of leaf tissue also increased from 300 to 500 mosmol kg^-1. These experimental conditions were considered suitable to study glycinebetaine biosynthesis and its induction by salinity in the absence of marked growth inhibition or metabolic disturbance. Radioactive labelled [¹⁴C]serine, ethanolamine and choline (all 1 mol, 13.3 MBq in 10 l) were fed to detached secondary leaves via the petiole 24 h after the exposure of plants to salt. The rate of isotope incorporation into water soluble products, lipids and residue was measured over a further 24 h. The major metabolic fate of exogenous [¹⁴C]choline and [¹⁴C]ethanolamine was incorporation into glycinebetaine while less ¹⁴C-label was found in phosphatidyl choline and phosphatidyl ethanolamine. Incorporation rates were identical in control and salinised leaves and were adequate to account for observed values of glycinebetaine accumulation previously reported in spinach. In contrast the labelling of glycinebetaine from [¹⁴C]serine was twice as great in salinated plants as in the controls. These results, together with short term labelling experiment with [¹⁴C]ethanolamine using leaf slices, were consistent with the formation of glycinebetaine via serine, ethanolamine and its methylated derivatives to choline with some control being exerted at the serine level. However a flux through the phosphorylated intermediates is not excluded.From a consideration of these results and the published data on barley subjected to water stress (Hanson and Scott, 1980 Plant Physiol. 66, 342–348) there appear to be significant differences in the biosynthetic pathways in spinach and barley.Abbreviations BHT butylated hydroxytoluerte (2,6-di-tert-butyl-4-methylphenol) - C₁ one-carbon fragment - 1,2DG diglyceride moiety - DW day weight - MCW methanol-chloroform-water (12:5:1, by vol.) - PA phosphatidic acid - PC phosphatidyl choline - PMME phosphatidyl monomethylethanolamine - PDME phosphatidyl dimethylethanolamine - PE phosphatidyl ethanolamine - PPO 2,5-diphenyloxazole - POPOP 1,4-bis(5-phenyloxazoyl) benzene     

Pyrrolizidine alkaloid biosynthesis,evolution of a pathway in plant secondary metabolism

The system of pyrrolizidine alkaloids has proven to be a powerful system for studying the evolution of a biosynthetic pathway in plant secondary metabolism. Pyrrolizidine alkaloids are typical plant secondary products produced by the plant as a defense against herbivores. The first specific enzyme, homospermidine synthase, has been shown to have evolved by duplication of the gene encoding deoxyhypusine synthase, which is involved in primary metabolism. Despite the identical function of homospermidine synthase for pyrrolizidine alkaloid biosynthesis in the various plant lineages, this gene duplication has occurred several times independently during angiosperm evolution. After duplication, these gene copies diverged with respect to gene function and regulation. In the diverse plant lineages producing pyrrolizidine alkaloids, homospermidine synthase has been shown to be expressed in a variety of tissues, suggesting that the regulatory elements were recruited individually after the duplication of the structural gene. The molecular, kinetic, and expression data of this system are discussed with respect to current models of gene and pathway evolution.     

Capsule biosynthesis and basic metabolism in Streptococcus pneumoniae are linked through the cellular phosphoglucomutase

Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae requires UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) for production of the [3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n) polymer. The generation of UDP-Glc proceeds by conversion of Glc-6-P to Glc-1-P to UDP-Glc and is mediated by a phosphoglucomutase (PGM) and a Glc-1-P uridylyltransferase, respectively. Genes encoding both a Glc-1-P uridylyltransferase (cps3U) and a PGM homologue (cps3M) are present in the type 3 capsule locus, but these genes are not essential for capsule production. In this study, we characterized a mutant that produces fourfold less capsule than the type 3 parent. The spontaneous mutation resulting in this phenotype was not contained in the type 3 capsule locus but was instead located in a distant gene (pgm) encoding a second PGM homologue. The function of this gene product as a PGM was demonstrated through enzymatic and complementation studies. Insertional inactivation of pgm reduced capsule production to less than 10% of the parental level. The loss of PGM activity in the insertion mutants also caused growth defects and a strong selection for isolates containing second-site suppressor mutations. These results demonstrate that most of the PGM activity required for type 3 capsule biosynthesis is derived from the cellular PGM.     

Rice OVATE family protein 6 regulates leaf angle by modulating secondary cell wall biosynthesis

Plant Molecular Biology - Secondary cell wall not only provides rigidity and mechanical resistance to plants, but also has a large impact on plant growth and adaptation to environments....     

Temporal control over cellular targeting through hybridization of folate-targeted dendrimers and PEG-PLA nanoparticles

Polymeric nanoparticles (NPs) and dendrimers are two major classes of nanomaterials that have demonstrated great potential for targeted drug delivery. However, their targeting efficacy has not yet met clinical needs, largely because of a lack of control over their targeting kinetics, which often results in rapid clearance and off-target drug delivery. To address this issue, we have designed a novel hybrid NP (nanohybrid) platform that allows targeting kinetics to be effectively controlled through hybridization of targeted dendrimers with polymeric NPs. Folate (FA)-targeted generation 4 poly(amidoamine) dendrimers were encapsulated into poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) NPs using a double emulsion method, forming nanohybrids with a uniform size (~100 nm in diameter) at high encapsulation efficiencies (69-85%). Targeted dendrimers encapsulated within the NPs selectively interacted with FA receptor (FR)-overexpressing KB cells upon release in a temporally controlled manner. The targeting kinetics of the nanohybrids were modulated using three different molecular weights (MW) of the PLA block (23, 30, and 45 kDa). The release rates of the dendrimers from the nanohybrids were inversely proportional to the MW of the PLA block, which dictated their binding and internalization kinetics with KB cells. Our results provide evidence that selective cellular interactions can be kinetically controlled by the nanohybrid design, which can potentially enhance targeting efficacy of nanocarriers.