Flavin-dependent thymidylate synthase: A novel pathway towards thymine

For several decades only one chemical pathway was known for the de novo biosynthesis of the essential DNA nucleotide, thymidylate. This reaction catalyzed by thyA or TYMS encoded thymidylate synthases is the last committed step in the biosynthesis of thymidylate and proceeds via the reductive methylation of uridylate. However, many microorganisms have recently been shown to produce a novel, flavin-dependent thymidylate synthase encoded by the thyX gene. Preliminary structural and mechanistic studies have shown substantial differences between these deoxyuridylate-methylating enzymes. Recently, both the chemical and kinetic mechanisms of FDTS have provided further insight into the distinctions between thyA and thyX encoded thymidylate synthases. Since FDTSs are found in several severe human pathogens their unusual mechanism offers a promising future for the development of antibiotic and antiviral drugs with little effect on human thymidylate biosynthesis.     

Cloning and sequencing of the gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough

Abstract The gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough (148 amino acid residues), the first flavoprotein for which a three-dimensional structure has been determined, was cloned with the use of two synthetic oligonucleotides, designed to recognize the coding sequence for amino acid residues 11–19 and 98–103, respectively. The two oligonucleotides were used to screen a library of 900 λ-clones of the D. vulgaris chromosome. A single clone, λFL1, reacting with both probes was isolated. The entire structural gene for flavodoxin is contained in the 15 kb insert of λFL1 as found by nucleic acid sequencing. The codon usage in the flavodoxin gene is strongly biased towards G or C in the third codon position. A table in which codon usage information from all genes of D. vulgaris sequenced to date is combined is presented and should facilitate further gene cloning with oligonucleotide probes.     

Structural Features of Human Monoamine Oxidase A Elucidated from cDNA and Peptide Sequences

Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neuropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO-A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C-terminal region. The presence of features characteristic of the ADP-binding fold suggests that the N-terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.     

Enzymes metabolizing dimethylamine, trimethylamine and trimethylamine N-oxide in the yeast Sporopachydermia cereana grown on amines as sole nitrogen source

Abstract Sporopachydermia cereana , an ascosporogenous yeast, grew on dimethylamine, trimethylamine or trimethylamine N -oxide as sole nitrogen sources and produced mono-oxygenases for dimethylamine and trimethylamine that were significantly more stable than the corresponding enzymes found in Candida utilis . No trimethylamine mono-oxygenase activity was found in S. cereana grown on dimethylamine. In cells grown on trimethylamine N -oxide (but not on the other nitrogen sources), evidence for an enzyme metabolizing the N -oxide, possibly an aldolase, but more probably a reductase was obtained. All these activities showed a similar requirement for the presence of FAD or FMN in the extract buffer during isolation to retain activity. Amine mono-oxygenase activities showed a similar sensitivity to inhibitors, including proadifen hydrochloride and carbon monoxide as the corresponding enzymes in C. utilis . The trimethylamine N -oxide-dependent oxidation of NADH was more sensitive to inhibition by EDTA, N -ethylmaleimide and β-phenylethylamine than the mono-oxygenases, and less sensitive to KCN, and activity was significantly higher with NADPH than was observed with the 2 mono-oxygenases.     

Evidence for two different blue-light-receptor systems for the fast responses of stimulation of photosynthetic capacity and acidification of the plant surface in brown algae

Two blue-light responses of Phaeophyta that are expressed within a few seconds of a blue-light stimulus were characterized with respect to their photoreception properties. The first response is the activation of red-light-saturated photosynthesis which can be stimulated to values up to 5 times the rates in red light, depending on the species. The second response is a blue-light-induced acidification measurable at the plant surface. Both responses have similar kinetic characteristics and thus led us initially to hypothesise that they were causally connected in the same transduction mechanism. The two responses have action spectra [measured for Ectocarpus siliculosus (Dillwyn) Lyngb. and Laminaria saccharina (L.) Lamouroux] that are indistinguishable within the relatively large limits of error. However, in all species tested, the threshold sensitivity for blue light of the photosynthetic response is lower than that of the pH-shift by a factor of 2 to 150. Furthermore, stimulation of photosynthesis is sensitive to the flavin inhibitors, KI and phenylacetic acid, but the pH response is not affected by these inhibitors. Thus, the blue-light-induced pH-shift does not cause the stimulation of photosynthesis. In contrast, the different fluence-response relationships of the two responses and particularly the differential effect of the inhibitors are clear evidence for the action of two independent transduction pathways and photoreceptor systems for blue light. At least photoreception for stimulation of photosynthesis involves a flavin-or and a pterin.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PAA phenylacetic acid We thank Dr. C. A. Maggs for collecting P. pavonica. This research was supported by National Environment Research Council grant No. GR3/8102.     

Absorption of the flavin dimers

The electronic absorption spectra of the flavomononucleotide (FMN) in aqueous solution and in glycerine-water solution with change of the dye concentration have been measured. The FMN dimer absorption spectrum, monomer absorption spectrum, dimerization constant K and molar fraction of the monomer were calculated. It was found that FMN dimerization constants in aqueous solution were K_a = 118.0 l/mol and in glycerine K_g = 20.5 l/mol. In the region of the monomer absorption band two dimer absorption bands appear, in accordance with the Kasha molecular exciton theory.     

Unveiling the Pathways of Dioxygen Through the C2 Component of the Environmentally Relevant Monooxygenase p‐Hydroxyphenylacetate Hydroxylase from Acinetobacter baumannii: A Molecular Dynamics Investigation

In this work, models of the homotetrameric C2 component of the monooxygenase p‐hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, in complex with dioxygen (O₂) and, or not, the substrate p‐hydroxyphenylacetate (HPA) were built. Both models proved to be amenable to random‐acceleration molecular dynamics (RAMD) simulations, whereby a tiny randomly oriented external force, acting on O₂ at the active site in front of flavin mononucleotide (FMNH^?), accelerated displacement of O₂ toward the bulk solvent. This allowed us to carry out a sufficiently large number of RAMD simulations to be of statistical significance. The two systems behaved very similarly under RAMD, except for O₂ leaving the active site more easily in the absence of HPA, but then finding similar obstacles in getting to the gate as when the active site was sheltered by HPA. This challenges previous conclusions that HPA can only reach the active center after that the C4aOOH derivative of FMNH^? is formed, requiring uptake of O₂ at the active site before HPA. According to these RAMD simulations, O₂ could well get to FMNH^? also in the presence of the substrate at the active site.     

The biochemistry of drug metabolism--an introduction: Part 2. Redox reactions and their enzymes

This review continues a general presentation of the metabolism of drugs and other xenobiotics started in a recent issue of Chemistry & Biodiversity. This Part 2 presents the numerous oxidoreductases involved, their nomenclature, relevant biochemical properties, catalytic mechanisms, and the very diverse reactions they catalyze. Many medicinally, environmentally, and toxicologically relevant examples are presented and discussed. Cytochromes P450 occupy a majority of the pages of Part 2, but a large number of relevant oxidoreductases are also considered, e.g., flavin-containing monooxygenases, amine oxidases, molybdenum hydroxylases, peroxidases, and the innumerable dehydrogenases/reductases.     

Selective ϖ-1 oxidation of fatty acids by CYP147G1 from Mycobacterium marinum

Background

Cyp147G1 is one of 47 cytochrome P450 encoding genes in Mycobacterium marinum M, a pathogenic bacterium with a high degree of sequence similarity to Mycobacterium tuberculosis and Mycobacterium ulcerans. Cyp147G1 is one of only two of these cyp genes which are closely associated with a complete electron transfer system.

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.