Low Biotin Concentrations Research Articles

The Wing of a Winged Helix-Turn-Helix Transcription Factor Organizes the Active Site of BirA, a Bifunctional Repressor/Ligase

The BirA biotin protein ligase of Escherichia coli belongs to the winged helix-turn-helix (wHTH) family of transcriptional regulators. The N-terminal BirA domain is required for both transcriptional regulation of biotin synthesis and biotin protein ligase activity. We addressed the structural and functional role of the wing of the wHTH motif in both BirA functions. A panel of N-terminal deletion mutant proteins including a discrete deletion of the wing motif were unable to bind DNA. However, all the N-terminal deletion mutants weakly complemented growth of a ΔbirA strain at low biotin concentrations, indicating compromised ligase activity. A wing domain chimera was constructed by replacing the BirA wing with the nearly isosteric wing of the E. coli OmpR transcription factor. Although this chimera BirA was defective in operator binding, it was much more efficient in complementation of a ΔbirA strain than was the wing-less protein. The enzymatic activities of the wing deletion and chimera proteins in the in vitro synthesis of biotinoyl-5'-AMP differed greatly. The wing deletion BirA accumulated an off pathway compound, ADP, whereas the chimera protein did not. Finally, we report that a single residue alteration in the wing bypasses the deleterious effects caused by mutations in the biotin-binding loop of the ligase active site. We believe that the role of the wing in the BirA enzymatic reaction is to orient the active site and thereby protect biotinoyl-5'-AMP from attack by solvent. This is the first evidence that the wing domain of a wHTH protein can play an important role in enzymatic activity.

Vhr1p, a New Transcription Factor from Budding Yeast, Regulates Biotin-dependent Expression of VHT1 and BIO5

Transcription of the Saccharomyces cerevisiae vitamin H transporter gene VHT1 is enhanced by low extracellular biotin. Here we present the identification and characterization of Vhr1p as a transcriptional regulator of VHT1 (VHR1 (YIL056w); VHT1 regulator 1) and the identification of the cis-regulatory target sequences for Vhr1p in two yeast promoters. VHR1 was identified in a complementation screening of mutagenized yeast cells that had lost the capacity to express the gene of the green fluorescent protein (GFP) from the VHT1 promoter. Deltavhr1 deletion mutants fail to induce VHT1 on low biotin concentrations. In yeast one-hybrid analyses performed with fusions of Vhr1p N-terminal and C-terminal fragments to the Gal4p activation domain or to the Gal4p DNA-binding domain, the Vhr1p N terminus mediated biotin-dependent DNA binding, and the Vhr1p C terminus triggered biotin-dependent transcriptional activation. The analyzed Vhr1p N-terminal fragment has previously been described as a domain of unknown function (DUF352). Deletion and linker scanning analyses of the VHT1 promoter revealed the palindromic 18-nucleotide sequence AATCA-N8-TGAYT as the vitamin H-responsive element. This sequence was identified also in the BIO5 promoter that is also transcriptionally activated on low biotin concentrations. Bio5p mediates the transport of 7-keto-8-aminopelargonic acid across the yeast plasma membrane, a compound that is used as a precursor in biotin biosynthesis. Deltavhr1 deletion mutants fail to induce BIO5 on low biotin concentrations. The presented data characterize Vhr1p as an essential component of the biotin-dependent signal transduction cascade in yeast.

Biotin Sensing in Saccharomyces cerevisiae Is Mediated by a Conserved DNA Element and Requires the Activity of Biotin-Protein Ligase

Biotin is a water-soluble vitamin that functions as a prosthetic group in carboxylation reactions. In addition to its role as a cofactor, biotin has multiple roles in gene regulation. We analyzed biotin effects on gene expression in the yeast Saccharomyces cerevisiae and demonstrated by microarray, Northern, and Western analyses that all yeast genes encoding proteins involved in biotin metabolism are up-regulated following biotin depletion. Many of these genes contain a palindromic promoter element that is necessary and sufficient for mediating the biotin response and functions as an upstream-activating sequence. Mutants lacking the plasma membrane biotin transporter Vht1p display constitutively high expression levels of biotin-responsive genes. However, they react normally to biotin precursors that do not require Vht1p for uptake. The biotin-like effect of precursors with regard to gene expression requires their intracellular conversion to biotin. This demonstrates that Vht1p does not act as a sensor for biotin and that intracellular biotin is crucial for gene expression. Mutants with defects in biotin-protein ligase, similar to vht1delta mutants, also display aberrantly high expression of biotin-responsive genes. Like vht1delta cells, they have reduced levels of protein biotinylation, but unlike vht1delta mutants, they possess normal levels of free intracellular biotin. This indicates that free intracellular biotin is irrelevant for gene regulation and identifies biotin-protein ligase as an important element of the biotin-sensing pathway in yeast.

Transport of Biotin in Human Keratinocytes

Biotin is an essential micronutrient for normal cellular function, growth, and development. Biotin deficiency leads to pathologic, dermatologic, and neurocutaneous manifestations in skin and its appendages. Previous studies described the presence of specific biotin transport systems in the epithelia of the intestine, liver, kidney, and placenta, and in blood mononuclear cells. The aim of this study was to examine biotin transport into human keratinocytes. Uptake of [3H]biotin was measured both in the HaCaT cell line and in native keratinocytes in primary culture. Uptake of [3H]biotin (6 nM) in HaCaT cells was linear for up to 5 min of incubation. In the presence of an Na+ gradient total biotin uptake was 4- to 5-fold higher than in the absence of sodium ions. Biotin uptake was not altered by H+ and Cl- gradients. This transport system exhibited a Michaelis-Menten constant for biotin of 22.7+/-1.0 microM and a maximal velocity of 163.6+/-3.5 pmol per 5 min per mg protein. [3H]Biotin uptake (6 nM) was strongly inhibited by lipoic acid (oxidized form, Ki=4.6 microM; reduced form, Ki=11.4 microM), pantothenic acid (Ki=1.2 microM), and desthiobiotin (Ki=15.2 microM), but not by biocytin or biotin methyl ester. Measured at [3H]biotin concentrations of 0.1-10 nM we obtained kinetic evidence for the presence of a second transport component that is saturable at very low biotin concentrations (Kt=2.6+/-0.1 nM). Unlabeled lipoic acid and pantothenic acid (20 nM) did not inhibit the [3H]biotin uptake (1 nM). We conclude that human keratinocytes express the Na+-dependent multivitamin transporter with preference for pantothenate and a very high affinity transport component with specificity for biotin.

Plant sucrose-H+ symporters mediate the transport of vitamin H.

A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake-deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP), suggesting an energy-dependent biotin-H+ symport mechanism. The transport activity could contribute to the so-far uncharacterized plant sucrose-H+ symporter AtSUC5 which mediates the energy-dependent transport of biotin and sucrose, and restores growth of the biotin transport-deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin-H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.

Plant sucrose‐H+ symporters mediate the transport of vitamin H

SummaryA cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake‐deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH‐group inhibitor p‐chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP), suggesting an energy‐dependent biotin‐H+ symport mechanism. The transport activity could contribute to the so‐far uncharacterized plant sucrose‐H+ symporter AtSUC5 which mediates the energy‐dependent transport of biotin and sucrose, and restores growth of the biotin transport‐deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin‐H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.

Identification of the Plasma Membrane H+-Biotin Symporter of Saccharomyces cerevisiae by Rescue of a Fatty Acid-auxotrophic Mutant

Bakers' yeast is auxotrophic for biotin (vitamin H) and depends on the efficient uptake of this compound from the environment. A mutant strain with strongly reduced biotin uptake and with reduced levels of protein biotinylation was identified. The strain was auxotrophic for long-chain fatty acids, and this auxotrophy could be suppressed with high levels of biotin in the medium. After transformation of this mutant with a yeast genomic library, the unassigned open reading frame YGR065C was identified to complement this mutation. This gene codes for a protein with 593 amino acids and 12 putative transmembrane helices. Northern blot analysis revealed that, in wild-type cells, the corresponding mRNA levels were increased at low biotin concentrations. Likewise, cellular biotin uptake was increased with decreasing biotin availability. Expression of YGR065C under the control of the constitutive ADH1 promoter resulted in very high biotin transport rates across the plasma membrane that were no longer regulated by the biotin concentration in the growth medium. We conclude that YGR065C encodes the first biotin transporter identified for a non-mammalian organism and designate this gene VHT1 for vitamin H transporter 1.

Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum.

The recent discovery that phosphoenolpyruvate carboxylase (PEPCx) is dispensable for growth and lysine production in Corynebacterium glutamicum implies that this organism possesses (an) alternative anaplerotic enzyme(s). In permeabilized cells of C. glutamicum, we detected pyruvate carboxylase (PCx) activity. This activity was effectively inhibited by low concentrations of ADP, AMP and acetyl-CoA. PCx activity was highest [45 ± 5 nmol min-1 (mg dry wt)-1] in cells grown on lactate or pyruvate, and was about two- to threefold lower when the cells were grown on glucose or acetate, suggesting that formation of PCx is regulated by the carbon source in the growth medium. In cells grown at low concentrations of biotin (< 5 μg I-1), PCx activity was drastically reduced, indicating that the enzyme is a biotin protein. Growth experiments with the wild-type and a defined PEPCx-negative mutant of C. glutamicum on glucose showed that the mutant has a significantly higher demand for biotin than the wild-type, whereas both strains have the same high biotin requirement for growth on lactate and the same low biotin requirement for growth on acetate. These results indicate that (i) PCx is an essential anaplerotic enzyme for growth on glucose in the absence of PEPCx, (ii) PCx is an essential anaplerotic enzyme for growth on lactate even in the presence of PEPCx, and (iii) PCx has no anaplerotic significance for growth on acetate as the carbon source. In support of these conclusions, screening for clones unable to grow on a minimal medium containing lactate, but able to grow on a medium containing glucose or acetate, led to the isolation of PCx-defective mutants of C. glutamicum.

Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin.

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2 x 10(-10) mol/l) or by pharmacological doses of biotin (10(-5) mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.

Biotin radioligand assay with an 125I-labeled biotin derivative, avidin, and avidin double-antibody reagents.

We describe a new radioligand assay for determining biotin in biological fluids by using a mixture of N-[beta-(4-OH-3-125I-phenyl)ethyl]- and N-[beta-(4-OH-3,5-di-125I-phenyl)ethyl]biotinamides as radiotracer, avidin as a binding protein, and an avidin double-antibody as a separation reagent. The radiotracer is synthesized by coupling (at pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to radioiodinated tyramine. The assay curve is linear and the assay itself is sensitive (less than 10 ng/L), reproducible (intra- and interassay CVs 4.1% and 7.0%, respectively), and allows the simultaneous handling of more than 100 samples in less than 4 h. Serum samples from apparently normal subjects contained 100-840 ng of biotin per liter (mean 340 ng/L). Pregnant women had low concentrations of biotin (100-300 ng/L) in their serum. Patients undergoing chronic hemodialysis treatment showed high concentrations (0.5-3.0 micrograms/L), which may be ascribable to the inability of avidin, which was used as the assay binding protein, to distinguish biotin from biotinyl derivatives with an intact ureido ring.

A carrier-mediated system for transport of biotin in rat intestine in vitro.

Transport of biotin was examined in rat intestine using the everted sac technique. Transport of 0.1 microM biotin was linear with time for at least 30 min of incubation and occurred at a rate of 3.7 pmol X g initial tissue wet wt-1 X min-1. Transport of biotin was higher in the jejunum than the ileum and was minimum in the colon (85 +/- 6, 36 +/- 6, and 2.8 +/- 0.6 pmol X g initial tissue wet wt-1 X 25 min-1, respectively). In the jejunum, transport of biotin was saturable at low concentrations (Kt = 3.73, microM, Vmax = 3.11 nmol X g initial tissue wet wt-1 X 25 min-1) but linear at higher concentrations (greater than 10 microM). The transport of low concentrations of biotin was inhibited by structural analogues (desthiobiotin, biotin methyl ester, diaminobiotin, and biocytin), Na+ dependent, energy dependent, temperature dependent, and proceeded against a concentration gradient in the serosal compartment. No metabolic alteration occurs to the biotin molecule during transport. This study demonstrates that biotin transport in rat intestine occurs by a carrier-mediated process at low concentrations and by simple diffusion at high concentrations. Furthermore, the carrier-mediated process is Na+, energy, and temperature dependent.

Biochemical evidence for diverse etiologies in biotin-responsive multiple carboxylase deficiency.

Biotin-responsive multiple carboxylase deficiency can be categorized by clinical criteria into a neonatal-onset disorder and distinct syndrome of infantile onset. Pedigrees in each instance are consistent with autosomal recessive inheritance. For a neonatal-onset proband, the sensitivity to relative biotin deprivation and the rapid clinical response to biotin supplementation are reflected by in vitro studies. Specific activities of biotin-dependent pyruvate carboxylase, propionyl CoA carboxylase, and 1-methylcrotonyl CoA carboxylase are 0.8 to 16% of mean control values after growth of fibroblasts in intermediate and very low biotin concentrations. Following relative biotin depletion, pyruvate carboxylase activity returns to normal after only 14 hr of growth in biotin-supplemented medium. In contrast, carboxylase activities in fibroblasts of an infantile-onset proband remain normal at very low biotin concentrations, even when avidin is added to the growth medium. The clinical heterogeneity, taken together with the distinct responses of cultured skin fibroblasts to biotin deprivation in vitro, probably reflect fundamentally different etiologies for the two categories of biotin-responsive multiple carboxylase deficiency.

NUTRITIONAL REQUIREMENTS OF THE MICROFLORA OF A SLIME ACCUMULATION IN A PAPER MILL

The nutrition of microorganisms isolated from an accumulation of slime has been examined. A number of the fungi and bacteria had an absolute requirement for one or more vitamins. The growth rates of others were markedly stimulated by low concentrations of biotin. In general, both mono- and disaccharides were readily utilized by the isolates. Geotrichum candidum, Candida reukaufii, and Sporotrichum sp. were unable to utilize nitrate nitrogen and several bacteria required amino acids.