Identification of cyclic intermediates in Azorhizobium caulinodans nicotinate catabolism.

In wild-type Azorhizobium caulinodans ORS571, nicotinate served both as anabolic substrate for NAD+ production and as catabolic substrate for use as the N source. Catabolic enzyme activities were greatest from cultures grown with nicotinate as the N source and least when cultures were grown with ammonium as the N source. Vector insertion mutants unable to catabolize nicotinate (nic::Vi mutants) still required micromolar quantities of this compound for growth. Therefore, A. caulinodans wild type is NAD+ auxotrophic. As the first two intermediates in A. caulinodans nicotinate catabolism, two cyclic compounds, 6-hydroxynicotinate and 1,4,5,6-tetrahydro-6-oxonicotinate, were identified. These compounds were purified from the growth medium of strain 61009 (a nic::Vi mutant) by high-performance liquid chromatography; their identities were subsequently confirmed by UV absorbance, nuclear magnetic resonance, and mass spectra. The conversion of 1 mol of nicotinate to 6-hydroxynicotinate consumed 0.5 mol of O2. From 18O isotopic incorporation experiments, water was the hydroxyl-equivalent source. A nicotinate hydroxylase activity proved to be cell wall-membrane associated; this activity served as direct electron donor (not indirect via NADP+) to O2 via membrane electron transport. These catabolic reactions have not previously been witnessed together in the same organism. A. caulinodans nicotinate catabolism seems coupled to N2 fixation, although the explicit mechanism of this coupling remains to be determined.     

Cloning of Azorhizobium caulinodans nicotinate catabolism genes and characterization of their importance in N2 fixation.

Twenty Azorhizobium caulinodans vector insertion (Vi) mutants unable to catabolize nicotinate (Nic- phenotype) were identified and directly cloned as pVi plasmids. These pVi plasmids were used as DNA hybridization probes to isolate homologous wild-type sequences. From subsequent physical mapping experiments, the nic::Vi mutants defined four distinct loci. Two, possibly three, of these loci are physically linked. A. caulinodans nic loci II and III encode the structural genes for nicotinate catabolism; nic loci I and IV encode nicotinate-driven respiratory chain components. Recombinant lambda bacteriophages corresponding to three of these loci were subcloned in pRK293; resulting plasmids were used for complementation tests with resolved nic::IS50 derivatives of the nic::Vi mutants. When wild-type A. caulinodans was cultured in defined liquid medium under 3% O2, nicotinate catabolism stimulated N2 fixation 10-fold. In these exponentially growing cultures, the entire (300 microM) nicotinate supplement was exhausted within 10 h. While nic::Vi mutants retained the ability to fix some N2, they did so at rates only 10% of that of the wild type: nitrogenase activity by nic::Vi mutants was not stimulated by 300 microM added nicotinate. Higher-level (5 mM) nicotinate supplementation inhibited N2 fixation. Because 5 mM nicotinate repressed nitrogenase induction in all nic::Vi mutants as well, this repression was independent of nicotinate catabolism. During catabolism, nicotinate is first oxidized to 6-OH-nicotinate by a membrane-bound nicotinate hydroxylase which drives a respiratory chain to O2. In A. caulinodans wild-type cultures, added 300 microM 6-OH-nicotinate stimulated N2 fixation twofold better than did added 300 microM nicotinate. Likewise, nic::Vi mutant 61302, defective in nicotinate hydroxylase, fixed N2 at wild-type levels when supplemented with 300 microM 6-OH-nicotinate. Therefore, nicotinate catabolism stimulates N2 fixation not by nicotinate hydroxylase-driven respiration but rather by some subsequent aspect(s) of nicotinate catabolism.     

Elucidation of the flavonoid catabolism pathway in Pseudomonas putida PML2 by comparative metabolic profiling.

Flavonoids are 15-carbon plant secondary metabolites exuded in the rhizosphere that hosts several flavonoid-degrading bacteria. We studied flavonoid catabolism in a plant growth-promoting rhizobacterial strain of Pseudomonas by using a combination of biochemical and genetic approaches. Transposants carrying mini-Tn5gfp insertions were screened for flavonoid auxotrophy, and these mutant strains were found to be unable to grow in the flavonols naringenin and quercetin, while their growth in glycerol was comparable to that of the parental strain. In order to understand flavonoid catabolism, culture supernatants, whole-cell fractions, cell lysate, and cell debris of the wild-type and mutant strains were analyzed. Intermediates that accumulated intracellularly and those secreted in the medium were identified by a combination of reversed-phase high-pressure liquid chromatography and electrospray ionization-mass spectrometry. Structures of four key intermediates were confirmed by one-dimensional nuclear magnetic resonance spectroscopy. Comparative metabolic profiling of the compounds in the wild-type and mutant strains allowed us to understand the degradation events and to identify six metabolic intermediates. The first step in the pathway involves 3,3'-didehydroxylation, followed by hydrolysis and cleavage of the C-ring, leading via subsequent oxidations to the formation of protocatechuate. This is the first report on quercetin dehydroxylation in aerobic conditions leading to naringenin accumulation.     

Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis

Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron‐chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non‐ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC‐MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl‐coenzyme A (CoA)‐hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)‐CoA synthase Hcs1 in U. maydis that HMG‐CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.     

Nicotinate catabolism is dispensable and nicotinate anabolism is crucial in Azorhizobium caulinodans growing in batch culture and chemostat culture on N2 as The N source.

When Azorhizobium caulinodans was grown in chemostat cultures with N2 as the N source at a constant dilution rate of 0.1 h-1 in media with a constant concentration (50 mM) of succinate and variable concentrations (1.5 to 585 microM) of nicotinate, neither the growth yield on succinate, the specific rate of O2 consumption, nor the specific rate of CO2 production showed linear regression with the concentration of nicotinate. Moreover, for transient continuous cultures in which the nicotinate concentration was gradually lowered, growth parameters remained unchanged until an apparently critical level of 0.7 microM nicotinate was reached. Below this nicotinate level, an immediate washout of the chemostat population began. A. caulinodans nicotinate hydroxylase-negative mutant 61007, unable to catabolize nicotinate, and the wild type behaved similarly. Thus, for continuous cultures supplied with N2 as the N source, submicromolar concentrations of nicotinate both sustained pyridine nucleotide biosynthesis at sufficient levels and precluded the use of nicotinate as a catabolic substrate. Furthermore, when more nicotinate was provided, dual succinate-nicotinate limitation in continuous cultures did not occur. Finally, when nicotinate is present in suboptimal concentrations, the specific growth rate is directly proportional to the amount of nicotinate present per unit of biomass. By contrast, in batch cultures with different nicotinate concentrations and with either succinate or lactate as the carbon and energy source, anomalous growth curves were obtained. With a low concentration (1.5 microM) of nicotinate, growth on N2 occurred, albeit at low rates. With a high concentration (195 microM) of nicotinate, growth on N2 was temporarily stimulated, but nicotinate was quickly exhausted and growth was thereafter nicotinate limited. Continuous supplementation of batch cultures with nicotinate allowed only transient exponential growth followed by linear growth. Thus, also for batch cultures, nicotinate catabolism is dispensable, although a high concentration of nicotinate temporarily stimulates growth on N2. Ut us concluded that A. caulinodans is a true diazotroph.     

The crystal structure of enamidase: a bifunctional enzyme of the nicotinate catabolism

The hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to 2-formylglutarate is a central step in the catabolism of nicotinate in several Clostridia and Proteobacteria. This reaction is catalyzed by the novel enzyme enamidase, a new member of the amidohydrolase superfamily as indicated by its unique reaction, sequence relationship, and the stoichiometric binding of iron and zinc. A hallmark of enamidase is its capability to catalyze a two-step reaction: the initial decyclization of 1,4,5,6-tetrahydro-6-oxonicotinate leading to 2-(enamine)glutarate followed by an additional hydrolysis step yielding (S)-2-formylglutarate. Here, we present the crystal structure of enamidase from Eubacterium barkeri at 1.9 Å resolution, providing a structural basis for catalysis and suggesting a mechanism for its exceptional activity and enantioselectivity. The enzyme forms a 222-symmetric tetramer built up by a dimer of dimers. Each enamidase monomer consists of a composite β-sandwich domain and an (α/β)₈-TIM-barrel domain harboring the active site. With its catalytic binuclear metal center comprising both zinc and iron ions, enamidase represents a special case of subtype II amidohydrolases.     

Elucidation of the melitidin biosynthesis pathway in pummelo

Specialized plant metabolism is a rich resource of compounds for drug discovery. The acylated flavonoid glycoside melitidin is being developed as an anti-cholesterol statin drug candidate, but its biosynthetic route in plants has not yet been fully characterized. Here, we describe the gene discovery and functional characterization of a new flavonoid gene cluster (UDP-glucuronosyltransferases (CgUGTs), 1,2 rhamnosyltransferase (Cg1,2RhaT), acyltransferases (CgATs)) that is responsible for melitidin biosynthesis in pummelo (Citrus grandis (L.) Osbeck). Population variation analysis indicated that the tailoring of acyltransferases, specific for bitter substrates, mainly determine the natural abundance of melitidin. Moreover, 3-hydroxy-3-methylglutaryl-CoA reductase enzyme inhibition assays showed that the product from this metabolic gene cluster, melitidin, may be an effective anti-cholesterol statin drug candidate. Co-expression of these clustered genes in Nicotiana benthamiana resulted in the formation of melitidin, demonstrating the potential for metabolic engineering of melitidin in a heterologous plant system. This study establishes a biosynthetic pathway for melitidin, which provides genetic resources for the breeding and genetic improvement of pummelo aimed at fortifying the content of biologically active metabolites.     

Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of Panax notoginseng using a synthetic biology platform

UDP-glycosyltransferase (UGT)-mediated glycosylation is a widespread modification of plant natural products (PNPs), which exhibit a wide range of bioactivities, and are of great pharmaceutical, ecological and agricultural significance. However, functional annotation is available for less than 2% of the family 1 UGTs, which currently has 20,000 members that are known to glycosylate several classes of PNPs. This low percentage illustrates the difficulty of experimental study and accurate prediction of their function. Here, a synthetic biology platform for elucidating the UGT-mediated glycosylation process of PNPs was established, including glycosyltransferases dependent on UDP-glucose and UDP-xylose. This platform is based on reconstructing the specific PNPs biosynthetic pathways in dedicated microbial yeast chassis by the simple method of plug-and-play. Five UGT enzymes were identified as responsible for the biosynthesis of the main glycosylation products of triterpenes in Panax notoginseng, including a novel UDP-xylose dependent glycosyltransferase enzyme for notoginsenoside R1 biosynthesis. Additionally, we constructed a yeast cell factory that yields >1 g/L of ginsenoside compound K. This platform for functional gene identification and strain engineering can serve as the basis for creating alternative sources of important natural products and thereby protecting natural plant resources.     

Intercellular pathway of leucine catabolism in rat spermatogenic epithelium.

A unique intercellular pathway of leucine catabolism was observed in vitro in rat spermatogenic epithelium. Sertoli cells convert leucine via transmination into 4-methyl-2-oxovalerate, and spermatocytes and spermatids reduce exogenous 4-methyl-2-oxovalerate to 2-hydroxy-4-methylvalerate, which is then released by the spermatogenic cells. The NADH-dependent reduction of 4-methyl-2-oxovalerate could be catalysed by the male-germ-cell-specific lactate dehydrogenase isoenzyme LDH-C4 in the cytosol of the spermatogenic cells, concomitant with the NAD+-dependent conversion of exogenous lactate into pyruvate.     

Elucidation of the beta-carotene hydroxylation pathway in Arabidopsis thaliana

The first dedicated step in plant xanthophyll biosynthesis is carotenoid hydroxylation. In Arabidopsis thaliana, this reaction is performed by both heme (LUT1 and LUT5) and non-heme (CHY1 and CHY2) hydroxylases. No mutant completely abolishing alpha- or beta-carotene hydroxylation has been described to date. We constructed double and triple mutant combinations in CHY1, CHY2, LUT1, LUT5 and LUT2 (lycopene epsilon-cyclase). In chy1chy2lut2, 80% of leaf carotenoids is represented by beta-carotene. In chy1chy2lut5, beta-carotene hydroxylation is completely abolished, while hydroxylation of the beta-ring of alpha-carotene is still observed. The data are consistent with a role of LUT5 in beta-ring hydroxylation, and with the existence of an additional hydroxylase, acting on the beta-ring of alpha-, but not beta-carotene.     

A complete pathway for β-alanine and β-amino-iso-butyrate catabolism in Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa PAO1 was found to catabolise β-alanine and β-amino- iso -butyrate (β-AIB) by the following pathway: (i) transamination by β-alanine: pyruvate aminotransferase (BAPAT) to yield l -alanine and either malonic semialdehyde or its methyl analogue, respectively; (ii) oxidative decarboxylation of the respective semialdehydes to acetyl CoA or propionyl CoA; (iii) regeneration of pyruvate from l -alanine by the action of dl -alanine racemase (AR) and d -alanine dehydrogenase (DAD). Mutants defective in BAPAT or DAD failed to catabolise either β-alanine or β-AIB, and β-alanine was an inducer for the entire pathway.     

Role of the ubiquitin-proteasome pathway in sepsis-induced muscle catabolism

Several lines of evidence suggest that the ubiquitin-proteasome pathway is involved in sepsis-induced muscle catabolism. The gene expression of ubiquitin and several of the proteasome subunits was increased in muscle from both septic rats and patients. In other studies, the activity of isolated 20S proteasomes was stimulated in septic muscles. Sepsis-induced increase in muscle total and myofibrillar protein breakdown was inhibited with specific proteasome blockers. Although the ubiquitin-proteasome pathway is upregulated in septic muscle, it is still unclear how the myofibrillar proteins actin and myosin are ubiquitinated and become substrates for the 26S proteasome. Recent studies suggest that a calcium-dependent, calpain-mediated process releases myofilaments from the Z-disks during sepsis. It is possible that this process exposes destabilizing N-terminal residues on actin and myosin, making them suitable substrates for the N-end rule pathway involving the 14 kD ubiquitin-conjugating enzyme E214k and the ubiquitin-protein ligase E3.     

Elucidation of the 2-aminoethylphosphonate biosynthetic pathway in Tetrahymena pyriformis

The biosynthetic reaction pathway leading to the natural product, 2-aminoethylphosphonate in Tetrahymena pyriformis has been elucidated. Incubation of [32P]PEP and [14C]PEP with T.pyriformis cellular homogenate fortified with Mg2+ and alanine/pyridoxal phosphate, yielded 2-aminoethylphosphonate as the minor reaction product (2-5% yield) and phosphoglycerate and pyruvate plus orthophosphate as the major products. Inclusion of thiamine pyrophosphate in the reaction mixture increased the yield of 2-aminoethylphosphonate by a factor of 10. Incubation of phosphonoacetaldehyde or phosphonopyruvate in the cellular homogenate also provided 2-aminoethylphosphonate. The cellular homogenate catalyzed the transformation of phosphonoacetaldehyde to 2-aminoethylphosphonate in an ca. 80% yield. However, the maximum yield of 2-aminoethylphosphonic acid obtained by use of phosphonopyruvate was only 15%. The major reaction pathways induced by treatment of phosphonopyruvate with the cellular extract involved its competitive conversion to PEP and pyruvate plus orthophosphate.     

Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway

Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.4 μg g⁻¹ parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding a costunolide and parthenolide 3β-hydroxylase was identified opening up further options to improve the water solubility of parthenolide and therefore its potential as a drug.     

A novel pathway for phosphatidylcholine catabolism in rat brain homogenates.

Rat brain homogenates incubated with exogenous [32-P] phosphatidylcholine liberated: LYSO[32-P] phosphatidylcholine, sn-glycero-3-[32-P] phosphorylcholine, [32-P] phosphorylcholine, sn-gleycero-3-[32-P] phosphate and 32-Pi. Further investigation showed that [32-P] phosphorylcholine was released exclusively from sn-glycero-3-[32-P] phosphorylcholien by a novel diesterase activity. We propose that the enzyme be termed L-3-glycerylphosphinicocholine cholinephosphohydrolase (EC 3.1.4.-). Parallel experiments on rat liver homogenates and a P815Y mouse mastocytoma cell-lysate, revealed no diesterase activity.