Identification of porphyrins produced from isopropanol by Arthrobacter hyalinus

When Arthrobacter hyalinus was grown on isopropanol, a large amount of red pigment was accumulated in the culture broth. The pigment was isolated from the culture broth. With thin layer chromatography, FD mass, IR, ¹H-NMR, ¹³C-NMR, and absorption spectra methods it was found that the red pigments were composed of type III varieties of coproporphyrin, penta carboxyl porphyrin, hexa carboxyl porphyrin, hepta carboxyl porphyrin and uroporphyrin, and some type I uroporphyrin.     

The biosynthetic pathway of new polyamines in Caldariella acidophila

A spontaneous mutant of Escherichia coli (strain AB2847), selected for resistance to the aminoglycoside antibiotic neamine, shows severe restriction of amber suppressors in vivo. Ribosomes isolated from the mutant exhibit only low misreading in vitro in the presence of the antibiotic. Genetic and biochemical analyses indicate that the neamine-resistant phenotype is the result of two distinct mutations. The first, res3128, appears to affect the gene (strA) coding for the ribosomal protein S12. Although it leads to a restrictive phenotype it does not, however, confer resistance to streptomycin. The second mutation, X3128, is located between the sirA and AROB loci and is lethal when segregated from the res3128 mutation. It may affect the ribosome at the level of a post-translational modification.     

A novel 4-hydroxycoumarin biosynthetic pathway

Coumarin forms in melilotoside (trans-ortho-coumaric acid glucoside)-containing plant species upon cell damage. In moldy melilotoside-containing plant material, trans-ortho-coumaric acid is converted by fungi to 4-hydroxycoumarin, two molecules of which spontaneously combine with formaldehyde to give dicoumarol. Dicoumarol causes internal bleeding in livestock and is the forerunner of the warfarin group of medicinal anticoagulants. Here, we report 4-hydroxycoumarin formation by biphenyl synthase (BIS). Two new BIS cDNAs were isolated from elicitor-treated Sorbus aucuparia cell cultures. The encoded isoenzymes preferred ortho-hydroxybenzoyl (salicoyl)-CoA as a starter substrate and catalyzed a single decarboxylative condensation with malonyl-CoA to give 4-hydroxycoumarin. When elicitor-treated S. aucuparia cell cultures were fed with the N-acetylcysteamine thioester of salicylic acid, 4-hydroxycoumarin accumulated in the culture medium. Incubation of the BIS isoenzymes with benzoyl-CoA and malonyl-CoA resulted in the formation of 3,5-dihydroxybiphenyl which is the precursor of the phytoalexins of the Maloideae.     

A C35 carotenoid biosynthetic pathway

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C(30) carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C(35) backbone. The products of condensation of farnesyldiphosphate and GDP, C(35) structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C(40) or C(30) pathways accepted and converted the C(35) substrate, thus creating a C(35) carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.     

Molecular characterization of the safracin biosynthetic pathway from Pseudomonas fluorescens A2-2: designing new cytotoxic compounds

Safracin is an antibiotic with anti-tumour activity produced by Pseudomonas fluorescens A2-2. The entire safracin synthetic gene cluster spanning 17.5 kb has been identified, cloned and sequenced. The safracin cluster comprises 10 open reading frames (ORFs) encoding proteins for three non-ribosomal peptide synthetases (NRPS), three safracin precursor biosynthetic enzymes, two safracin tailoring enzymes, a safracin resistance protein and a small hypothetical protein of unknown function. These genes are organized in two divergent operons of eight and two genes respectively. This pathway exhibits unusual features when compared with other NRPS systems. We have demonstrated by heterologous expression of the cluster that it is able to direct the synthesis of safracin in other strains. Cross-feeding experiments have confirmed that 3-hydroxy-5-methyl-O-methyltyrosine is the precursor of two amino acids of the molecule. Genetic analyses have allowed us to demonstrate that the bicistronic operon encodes the hydroxylation and N-methylation activities of the pathway. The cloning and expression of the safracin cluster has settled the basis for the in vivo and in vitro production of a wide variety of compounds, such as the promising ecteinascidins anti-cancer compounds.     

A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation

Ero1 and Pdi1 are essential elements of the pathway for the formation of disulphide bonds within the endoplasmic reticulum (ER). By screening for alternative oxidation pathways in Saccharomyces cerevisiae, we identified ERV2 as a gene that when overexpressed can restore viability and disulphide bond formation to an ero1-1 mutant strain. ERV2 encodes a luminal ER protein of relative molecular mass 22,000. Purified recombinant Erv2p is a flavoenzyme that can catalyse O2-dependent formation of disulphide bonds. Erv2p transfers oxidizing equivalents to Pdi1p by a dithiol-disulphide exchange reaction, indicating that the Erv2p-dependent pathway for disulphide bond formation closely parallels that of the previously identified Ero1p-dependent pathway.     

Drosophila yellow-h encodes dopaminechrome tautomerase: A new enzyme in the eumelanin biosynthetic pathway

Melanin is a widely distributed phenolic pigment that is biosynthesized from tyrosine and its hydroxylated product, dopa, in all animals. However, recent studies reveal a significant deviation from this paradigm, as insects appear to use dopamine rather than dopa as the major precursor of melanin. This observation calls for a reconsideration of the insect melanogenic pathway. While phenoloxidases and laccases can oxidize dopamine for dopaminechrome production, the fate of dopaminechrome remains undetermined. Dopachrome decarboxylase/tautomerase, encoded by yellow-f/f2 of Drosophila melanogaster, can convert dopaminechrome into 5,6-dihydroxyindole, but the same enzyme from other organisms does not act on dopaminechrome, suggesting the existence of a specific dopaminechrome tautomerase (DPT). We now report the identification of this novel enzyme that biosynthesizes 5,6-dihydroxyindole from dopaminechrome in Drosophila. Dopaminechrome tautomerase acted on both dopaminechrome and N-methyl dopaminechrome but not on dopachrome or other aminochromes tested. Our biochemical and molecular studies reveal that this enzyme is encoded by the yellow-h gene, a member of the yellow gene family, and advance our understanding of the physiological functions of this gene family. Identification and characterization of DPT clarifies the precursor for melanin biosynthetic pathways and proves the existence of an independent melanogenic pathway in insects that utilizes dopamine as the primary precursor.     

Novel biosynthetic pathway of castasterone from cholesterol in tomato

Endogenous brassinosteroids (BRs) in tomato (Lycopersicon esculentum) seedlings are known to be composed of C27- and C28-BRs. The biosynthetic pathways of C27-BRs were examined using a cell-free enzyme solution prepared from tomato seedlings that yielded the biosynthetic sequences cholesterol --> cholestanol and 6-deoxo-28-norteasterone <--> 6-deoxo-28-nor-3-dehydroteasterone <--> 6-deoxo-28-nortyphasterol --> 6-deoxo-28-norcastasterone --> 28-norcastasterone (28-norCS). Arabidopsis CYP85A1 that was heterologously expressed in yeast mediated the conversion of 6-deoxo-28-norCS to 28-norCS. The same reaction was catalyzed by an enzyme solution from wild-type tomato but not by an extract derived from a tomato dwarf mutant with a defect in CYP85. Furthermore, exogenously applied 28-norCS restored the abnormal growth of the dwarf mutant. These findings indicate that the C-6 oxidation of 6-deoxo-28-norCS to 28-norCS in tomato seedlings is catalyzed by CYP85, just as in the conversion of 6-deoxoCS to CS. Additionally, the cell-free solution also catalyzed the C-24 methylation of 28-norCS to CS in the presence of NADPH and S-adenosylmethionine (SAM), a reaction that was clearly retarded in the absence of NADPH and SAM. Thus it seems that C27-BRs, in addition to C28-BRs, are important in the production of more active C28-BRs and CS, where a SAM-dependent sterol methyltransferase appears to biosynthetically connect C27-BRs to C28-BRs. Moreover, the tomato cell-free solution converted CS to 26-norCS and [2H6]CS to [2H3]28-norCS, suggesting that C-28 demethylation is an artifact due to an isotope effect. Although previous feeding experiments employing [2H6]CS suggested that 28-norCS was synthesized from CS in certain plant species, this is not supported in planta. Altogether, this study demonstrated for the first time, to our knowledge, that 28-norCS is not synthesized from CS but from cholesterol. In addition, CS and [2H6]CS were not converted into BL and [2H6]BL, respectively, confirming an earlier finding that the active BR in tomato seedlings is not BL but CS. In conclusion, the biosynthesis of 28-norBRs appears to play a physiologically important role in maintaining homeostatic levels of CS in tomato seedlings.     

The structure of two N-methyltransferases from the caffeine biosynthetic pathway

Caffeine (1,3,7-trimethylxanthine) is a secondary metabolite produced by certain plant species and an important component of coffee (Coffea arabica and Coffea canephora) and tea (Camellia sinensis). Here we describe the structures of two S-adenosyl-l-methionine-dependent N-methyltransferases that mediate caffeine biosynthesis in C. canephora 'robusta', xanthosine (XR) methyltransferase (XMT), and 1,7-dimethylxanthine methyltransferase (DXMT). Both were cocrystallized with the demethylated cofactor, S-adenosyl-L-cysteine, and substrate, either xanthosine or theobromine. Our structures reveal several elements that appear critical for substrate selectivity. Serine-316 in XMT appears central to the recognition of XR. Likewise, a change from glutamine-161 in XMT to histidine-160 in DXMT is likely to have catalytic consequences. A phenylalanine-266 to isoleucine-266 change in DXMT is also likely to be crucial for the discrimination between mono and dimethyl transferases in coffee. These key residues are probably functionally important and will guide future studies with implications for the biosynthesis of caffeine and its derivatives in plants.     

The molecular architecture of major enzymes from ajmaline biosynthetic pathway

The biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have been performed and their catalytic residues have been identified. Success with over-expression of the enzymes was an important step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed β-propeller fold the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this fold. The structure of strictosidine glucosidase was determined and its structure has as a (β/α)₈ barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield new alkaloid.     

Determining the structure of a biosynthetic pathway from incorporation measurements

Incorporation data, collected judiciously from a biosynthetic tracer experiment, can be processed to yield a considerable body of detailed information     

Tetrahydrobiopterin biosynthetic pathway and deficiency

It has been proven that the most common defect in the tetrahydrobiopterin biosynthesis is caused by 6-pyruvoyl tetrahydropterin synthase deficiency. The enzyme 6-pyruvoyl tetrahydropterin synthase consists of four identical subunits which convert dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin in the presence of magnesium. UV, NMR, and MS data prove that the enzyme catalyzes the elimination of triphosphate as well as the intramolecular rearrangement. The 6-pyruvoyl tetrahydropterin synthase activity was measured in fetal erythrocytes and together with the neopterin and biopterin measurements in amniotic fluid this enabled performing prenatal diagnosis of 6-pyruvoyl tetrahydropterin synthase deficiency. Peripheral tetrahydrobiopterin deficiency was shown to be due to an incomplete 6-pyruvoyl tetrahydropterin synthase deficiency or heterozygosity.     

The final player in the coenzyme A biosynthetic pathway

The determination of the crystal structure of human phosphopantothenoylcysteine synthetase completes our knowledge of the enzyme structures involved in all steps of coenzyme A biosynthesis. This structure provides insight into the differences between bacterial and mammalian forms of the enzyme and may guide the structure-based development of novel antibacterial compounds.     

Pyrimidine biosynthetic pathway of Pseudomonas fluorescens

Pyrimidine biosynthesis in Pseudomonas fluorescens strain A126 was investigated. In this study, de novo pyrimidine biosynthetic pathway mutant strains were isolated using both conventional mutagenesis and transposon mutagenesis. The resulting mutant strains were deficient for either aspartate transcarbamoylase, dihydroorotase or orotate phosphoribosyltransferase activity. Uracil, uridine or cytosine could support the growth of every mutant strain selected. In addition, the aspartate transcarbamoylase mutant strains could utilize orotic acid to sustain their growth while the orotidine-5'-monophosphate decarboxylase mutant strains grew slowly upon uridine 5'-monophosphate. The wild-type strain and the mutant strains were used to study possible regulation of de novo pyrimidine biosynthesis in P. fluorescens. Dihydroorotase specific activity more than doubled after the wild-type cells were grown in orotic acid relative to unsupplemented minimal-medium-grown cells. Starving the mutant strains of pyrimidines also influenced the levels of several de novo pyrimidine biosynthetic pathway enzyme activities.     

A dopamine-based biosynthetic pathway produces decarboxylated betalains in Chenopodium quinoa

Betalains are the nitrogenous pigments that replace anthocyanins in the plant order Caryophyllales. Here, we describe unconventional decarboxylated betalains in quinoa (Chenopodium quinoa) grains. Decarboxylated betalains are derived from a previously unconsidered activity of the 4,5-DOPA-extradiol-dioxygenase enzyme (DODA), which has been identified as the key enzymatic step in the established biosynthetic pathway of betalains. Here, dopamine is fully characterized as an alternative substrate of the DODA enzyme able to yield an intermediate and structural unit of plant pigments: 6-decarboxy-betalamic acid, which is proposed and described. To characterize this activity, quinoa grains of different colors were analyzed in depth by chromatography, time-of-flight mass spectrometry, and reactions were performed in enzymatic assays and bioreactors. The enzymatic-chemical scheme proposed leads to an uncharacterized family of 6-decarboxylated betalains produced by a hitherto unknown enzymatic activity. All intermediate compounds as well as the final products of the dopamine-based biosynthetic pathway of pigments have been unambiguously determined and the reactions have been characterized from the enzymatic and functional perspectives. Results evidence a palette of molecules in quinoa grains of physiological relevance and which explain minor betalains described in plants of the Caryophyllales order. An entire family of betalains is anticipated.

A biosynthetic pathway produces unconventional plant pigments betalains derived from dopamine in quinoa.