The biosynthesis of biotin in growing yeast cells: The formation of biotin from an early intermediate.
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ABSTRACT:
SUBMITTER: Eisenberg MA
PROVIDER: S-EPMC1270159 | biostudies-other | 1966 Dec
REPOSITORIES: biostudies-other
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Project description:1. An unknown biotin vitamer was obtained in high yields in culture filtrates of Penicillium chrysogenum. 2. Production of this vitamer and desthiobiotin is controlled by the biotin concentration in the medium. 3. The unknown vitamer becomes labelled when the organism is grown in the presence of radioactive pimelic acid. 4. Chromatographic procedures were developed for the purification of the radioactive vitamer. 5. The vitamer is extremely stable in concentrated acid but gives rise to new vitamers under certain conditions. 6. The intermediate role of this vitamer in the synthesis of biotin is discussed.
| S-EPMC1270160 | biostudies-other
Project description:Biotin synthase catalyzes the conversion of dethiobiotin (DTB) to biotin through the oxidative addition of sulfur between two saturated carbon atoms, generating a thiophane ring fused to the existing ureido ring. Biotin synthase is a member of the radical SAM superfamily, composed of enzymes that reductively cleave S-adenosyl-l-methionine (SAM or AdoMet) to generate a 5'-deoxyadenosyl radical that can abstract unactivated hydrogen atoms from a variety of organic substrates. In biotin synthase, abstraction of a hydrogen atom from the C9 methyl group of DTB would result in formation of a dethiobiotinyl methylene carbon radical, which is then quenched by a sulfur atom to form a new carbon-sulfur bond in the intermediate 9-mercaptodethiobiotin (MDTB). We have proposed that this sulfur atom is the μ-sulfide of a [2Fe-2S](2+) cluster found near DTB in the enzyme active site. In the present work, we show that formation of MDTB is accompanied by stoichiometric generation of a paramagnetic FeS cluster. The electron paramagnetic resonance (EPR) spectrum is modeled as a 2:1 mixture of components attributable to different forms of a [2Fe-2S](+) cluster, possibly distinguished by slightly different coordination environments. Mutation of Arg260, one of the ligands to the [2Fe-2S] cluster, causes a distinctive change in the EPR spectrum. Furthermore, magnetic coupling of the unpaired electron with (14)N from Arg260, detectable by electron spin envelope modulation (ESEEM) spectroscopy, is observed in WT enzyme but not in the Arg260Met mutant enzyme. Both results indicate that the paramagnetic FeS cluster formed during catalytic turnover is a [2Fe-2S](+) cluster, consistent with a mechanism in which the [2Fe-2S](2+) cluster simultaneously provides and oxidizes sulfide during carbon-sulfur bond formation.
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Project description:In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections.
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Project description:The effect of administration of biotin to biotin-deficient rats on protein biosynthesis was studied. Biotin treatment resulted in stimulation by more than twofold of amino acid incorporation into protein, both in vivo and in vitro in rat liver, pancreas, intestinal mucosa and skin. Analysis of the products of amino acid incorporation into liver proteins in vivo and in vitro indicated that the synthesis of some proteins was stimulated more than twofold, but others were not stimulated at all. This indicates a specificity in the stimulation of protein synthesis mediated by biotin.
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Project description:Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes.
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Project description:The ai5γ group II intron requires a protein cofactor to facilitate native folding in the cell. Yeast protein Mss116 greatly accelerates intron folding under near-physiological conditions both in vivo and in vitro. Although the effect of Mss116 on the kinetics of ai5γ ribozyme folding and catalysis has been extensively studied, the precise structural role and interaction sites of Mss116 have been elusive. Using Nucleotide Analog Interference Mapping to study the folding of splicing precursor constructs, we have identified specific intron functional groups that participate in Mss116-facilitated folding and we have determined their role in the folding mechanism. The data indicate that Mss116 stabilizes an early, obligate folding intermediate within intron domain 1, thereby laying the foundation for productive folding to the native state. In addition, the data reveal an important role for the IBS2 exon sequence and for the terminus of domain 6, during the folding of self-splicing group IIB intron constructs.
| S-EPMC4419380 | biostudies-literature
Project description:In eukaryotic organisms, Elongator is a six-subunit protein complex required for the formation of 5-carbamoylmethyl (ncm(5) ) and 5-methylcarboxymethyl (mcm(5) ) side chains on uridines present at the wobble position (U34 ) of tRNA. The open reading frame encoding the largest Elongator subunit Elp1p has two in-frame 5' AUG methionine codons separated by 48 nucleotides. Here, we show that the second AUG acts as the start codon of translation. Furthermore, Elp1p was previously shown to exist in two major forms of which one was generated by proteolysis of full-length Elp1p and this proteolytic cleavage was suggested to regulate Elongator complex activity. In this study, we found that the vacuolar protease Prb1p was responsible for the cleavage of Elp1p. The cleavage occurs between residues 203 (Lys) and 204 (Ala) as shown by amine reactive Tandem Mass Tag followed by LC-MS/MS (liquid chromatography mass spectrometry) analysis. However, using a modified protein extraction procedure, including trichloroacetic acid, only full-length Elp1p was observed, showing that truncation of Elp1p is an artifact occurring during protein extraction. Consequently, our results indicate that N-terminal truncation of Elp1p is not likely to regulate Elongator complex activity.
| S-EPMC4694139 | biostudies-literature
Project description:BackgroundTranslation of messenger mRNAs makes significant contributions to the control of gene expression in all eukaryotes. Because translational control often involves fractional changes in translational activity, good quantitative descriptions of translational activity will be required to achieve a comprehensive understanding of this aspect of biology. Data on translational activity are difficult to generate experimentally under physiological conditions, however, translational activity as a parameter is in principle accessible through published genome-wide datasets.ResultsAn examination of the accuracy of genome-wide expression datasets generated for Saccharomyces cerevisiae shows that the available datasets suffer from large random errors within studies as well as systematic shifts in reported values between studies, which make predictions of translational activity at the level of individual genes relatively inaccurate. In contrast, predictions of cell-wide translational activity are possible from such datasets with higher accuracy, and current datasets predict a production rate of about 13,000 proteins per haploid cell per second under fast growth conditions. This prediction is shown to be consistent with independently derived kinetic information on nucleotide exchange reactions that occur during translation, and on the ribosomal content of yeast cells.ConclusionThis study highlights some of the limitations in published genome-wide expression datasets, but also demonstrates a novel use for such datasets in examining global properties of cells. The global translational activity of yeast cells predicted in this study is a useful benchmark against which biochemical data on individual translation factor activities can be interpreted.
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Project description:Biotin (vitamin B7) is an enzyme cofactor required by organisms from all branches of life but synthesized only in microbes and plants. In the final step of biotin biosynthesis, a radical S-adenosyl-l-methionine (SAM) enzyme, biotin synthase (BioB), converts the substrate dethiobiotin to biotin through the stepwise formation of two C-S bonds. Previous electron paramagnetic resonance (EPR) spectroscopic studies identified a semistable intermediate in the formation of the first C-S bond as 9-mercaptodethiobiotin linked to a paramagnetic [2Fe-2S] cluster through one of its bridging sulfides. Herein, we report orientation-selected pulse EPR spectroscopic results that reveal hyperfine interactions between the [2Fe-2S] cluster and a number of magnetic nuclei (e.g., 57Fe, 15N, 13C, and 2H) introduced in a site-specific manner via biosynthetic methods. Combining these results with quantum chemical modeling gives a structural model of the intermediate showing that C6, the target of the second hydrogen-atom abstraction, is now in close proximity to the nascent thioether sulfur and is ideally positioned for the second C-S bond forming event.
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Project description:It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108. The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 A in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction. These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule. Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.
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