Clinical and biochemical consequences of p450 oxidoreductase deficiency

Patients with P450 oxidoreductase (POR) deficiency typically present with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley-Bixler craniosynostosis syndrome. Since our first report in 2004, more than 40 POR mutations have been identified in over 65 patients. POR is the obligate electron donor to all microsomal P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46, XY newborns as well as overvirilization in those who are 46, XX. This may be explained by impaired aromatization of fetal androgens that may cause maternal virilization and low urinary estriol levels during pregnancy. In addition, the alternate 'backdoor' pathway of androgen biosynthesis, which leads to dihydrotestosterone production bypassing androstenedione and testosterone, may also play a role. Functional assays studying the effects of POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. POR variants may also affect skeletal development and drug metabolism. As most drugs are metabolized by hepatic microsomal P450 enzymes, studies of the impact of POR mutations on drug-metabolizing P450s are particularly important.

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia

Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

Modeling of human P450 oxidoreductase structure by in silico mutagenesis and MD simulation

P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450s and mutations in POR cause several metabolic disorders. We have modeled the structure of human P450 oxidoreductase by in silico amino acid replacements in the rat POR crystal structure. The rat POR has 94% homology with human POR and 38 amino acids were replaced to make its sequence identical to human POR. Several rounds of molecular dynamic simulations refined the model and removed structural clashes from side chain alterations of replaced amino acids. This approach has the advantage of keeping the cofactor contacts and structural features of the core enzyme intact which could not be achieved by homology based approaches. The final model from our approach was of high quality and compared well with experimentally determined structures of other PORs. This model will be used for analyzing the structural implications of mutations and polymorphisms in human POR.

Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome

Deficient activities of multiple steroidogenic enzymes have been reported without and with Antley-Bixler syndrome (ABS), but mutations of corresponding cytochrome P450 enzymes have not been found. We identified mutations in POR, encoding P450 oxidoreductase, the obligate electron donor for these enzymes, in a woman with amenorrhea and three children with ABS, even though knock-out of POR is embryonically lethal in mice. Mutations of POR also affect drug-metabolizing P450 enzymes, explaining the association of ABS with maternal fluconazole ingestion.

In situ dehydration behavior of veszelyite (Cu,Zn)2Zn(PO4)(OH)3·2H2O: A single-crystal X-ray study

The rare mixed copper-zinc phosphate mineral veszelyite (Cu,Zn)2Zn(PO4)(OH)3·2H2O space group P21/c, a = 7.5096(2), b = 10.2281(2), c = 9.8258(2) Å, β = 103.3040(10)°, V = 734.45(3) Å3 was investigated by in situ temperature-dependent single-crystal X-ray structure refinements. The atomic arrangement of veszelyite consists of an alternation of octahedral and tetrahedral sheets. The Jahn-Teller distorted CuO6 octahedra form sheets with eight-membered rings. The tetrahedral sheet composed of PO4 and ZnO3(OH) tetrahedra shows strong topological similarities to that of cavansite, gismondine, and kipushite.Diffraction data of a sample from Zdravo Vrelo, near Kreševo (Bosnia and Herzegovina) have been measured in steps of 25 up to 225 °C. Hydrogen positions and the hydrogen-bond system were determined experimentally from the structure refinements of data collected up to 125 °C. At 200 °C, the hydrogen-bonding scheme was inferred from bond-valence calculations and donor-acceptor distances. The hydrogen-bond system connects the tetrahedral sheet to the octahedral sheet and also braces the Cu sheet.At 150 °C, the H2O molecule at H2O2 was released and the Cu coordination (Cu1 and Cu2) decreased from originally six- to fivefold. Cu1 has a square planar coordination by four OH groups and an elongate distance to O3, whereas Cu2 has the Jahn-Teller characteristic elongate bond to H2O1. The unit-cell volume decreased 7% from originally 734.45(3) to 686.4(4) Å3 leading to a formula with 1 H2O pfu. The new phase observed above 150 °C is characterized by an increase of the c axis and a shortening of the b axis. The bending of T-O-T angles causes an increasing elliptical shape of the eight-membered rings in the tetrahedral and octahedral sheets. Moreover a rearrangement of the hydrogen-bond system was observed.At 225 °C, the structure degrades to an X-ray amorphous residual due to release of the last H2O molecule at H2O1. The stronger Jahn-Teller distortion of Cu1 relative to Cu2 suggests that Cu1 is fully occupied by Cu, whereas Cu2 bears significant Zn. H2O1 is the fifth ligand of Cu2. Zn at Cu2 is not favorable to adopt planar fourfold coordination. Thus, if the last water molecule is expelled the structure is destabilized.This study contributes to understanding the dehydration mechanism and thermal stability of supergene minerals characterized by Jahn-Teller distorted octahedra with mixed Cu, Zn occupancy.

The role of menaquinone in the nitrate reductase complex

Membrane bound, respiratory nitrate reductase in Escherichia coli is composed of three subunits, αβγ. The active complex is anchored to the membrane by membrane-integrated γ subunit and can reduce nitrate to nitrite with membrane quinones, (ubiquinone or menaquinone) as physiological electron donors. The transfer of electrons through the complex is thought to involve the sequence: membrane quinols → b-type hemes (γ subunit) → Fe-S centers (β subunit) → molybdopterin (α subunit) → nitrate. The enzyme can be assayed with the artificial electron donor reduced methyl viologen (MVH) which transfers electrons directly to the molybdopterin cofactor. These studies have focused on the possible role of protein-bound menaquinone in the structure and function of this multisubunit complex. ^ Nitrate reductase was purified as two distinct forms; after solubilization of membrane proteins with detergents, purification rendered an αβγ complex (holoenzyme) which catalyzes nitrate reduction with MVH or the quinols analogs, menadiol and duroquinol, as electron donors. Alternatively, heat-treatment of the membranes in the absence of detergents and subsequent purification of the active enzyme produced an αβ complex, which reduces nitrate only with MVH as electron donor. The active αβ dimer was also separated from γ subunit by heat treatment of the holoenzyme. ^ Menaquinone-9 was isolated directly from the purified αβ complex, and identified by mass spectrometry. Based on the composition of the membrane quinone pool, it was concluded that menaquinone-9 is sequestered from the membrane pool in a specifically protein-bound form. ^ The role of the bound menaquinone in the structure-function of nitrate reductase was also investigated, along with its participation in UV-light inactivation of the enzyme. Menaquinone-depleted nitrate reductase from a menaquinone deficient mutant retained activity with all electron donors and it remained sensitive to UV inactivation. However, the MVH-nitrate reductase activity and the rate of UV inactivation of the enzyme were significantly reduced and the optical properties of the enzyme were modified by the absence of the bound menaquinone-9. ^ Menaquinone-9 is not absolutely required for electron transfer in nitrate reductase but it appears to be specifically-bound during assembly of the complex and to enhance the transfer of electrons through the complex. The possible plasticity of the functional electron transfer pathway in nitrate reductase is discussed. ^

Efficient and Robust Host–Guest Antenna Composite for Light Harvesting

We report discovery of a new efficient and robust antenna composite for light harvesting. The organic dye hostasol red (HR) is strongly luminescent in aprotic solvents but only weakly luminescent in potassium zeolite L (ZL) at ambient conditions. We observed a dramatic increase of the luminescence quantum yield of HR–ZL composites if some or all exchangeable potassium cations of ZL are substituted by an organic imidazolium cation (IMZ+) and if the acceptor HR is embedded in the middle part of the channels, so that it is fully protected by the environment of the perylene dye tb-DXP. This led to the discovery of a highly efficient donor,acceptor-ZL antenna material where tb-DXP acts as donor and HR acts as acceptor. The material has a donor-to-acceptor (D/A) absorption ratio of more than 100:1 and a nearly quantitative FRET efficiency. Synthesis of this host–guest material is reported. We describe a successful procedure for achieving full sealing of the ZL channel entrances such that the guests cannot escape. This new material is of great interest for applications in luminescent solar concentrator (LSC) devices because the efficiency killing self-absorption is very low.

Hydrogen-bonding complexes of functionalized BEDT-TTF derivatives with chloranilic acid

The two crystalline donor-acceptor complexes showing hydrogen-bondings between bis(ethylenedithio) tetrathiofulvalene (BEDT-TTF) derivatives containing pyridine and pyrazine groups and 2,5-dichloro-3,6-dihydroxyl-1,4-benzoquinone (chloranilic acid) were prepared. X-ray structure analyses revealed that functional groups play an important role in constructing the unique crystal structures.

Coordination-directed self-assembly of a simple benzothiadiazole-fused tetrathiafulvalene to low-bandgap metallogels

Coordination-driven gelation of a benzothiadiazole-fused tetrathiafulvalene (TTF) is demonstrated. This is the first work reporting highly stable metallogels based on a donor-acceptor conjugate with such a simple structure for the construction of new low-bandgap materials with various functional properties and novel nanostructures.

Dissolved lithium and silicon through time in microbial dissolution experiments on ODP Leg 209 peridotite

Using peridotite drilled during Ocean Drilling Program Leg 209, a series of enrichment cultures were initiated on board the ship to stimulate microbially enhanced dissolution of olivine. Dissolution was estimated by measured changes in dissolved Li and Si in the media through time (up to 709 days). The results suggest that there was no significant difference between the amounts of dissolved Li and Si in most of the inoculated microbial cultures compared to the control cultures. Alternative explanations for this are that 1. No microbes are living in the culture tubes that can affect the dissolution rates of olivine, 2. The control cultures have microbes effecting the dissolution of olivine as well as the inoculated cultures, 3. Not enough time has passed to build up a large enough microbial population to effect the dissolution of the olivine in the culture tubes, 4. Microbes act to suppress dissolution of olivine instead of enhancing dissolution, and 5. Abiotic dissolution overshadows microbially enhanced dissolution. Further work is required to test these alternatives.

Organic geochemistry of deep sea sediments from the Nankai Trough and the Southwest Japan forearc region

Earth's largest reactive carbon pool, marine sedimentary organic matter, becomes increasingly recalcitrant during burial, making it almost inaccessible as a substrate for microorganisms, and thereby limiting metabolic activity in the deep biosphere. Because elevated temperature acting over geological time leads to the massive thermal breakdown of the organic matter into volatiles, including petroleum, the question arises whether microorganisms can directly utilize these maturation products as a substrate. While migrated thermogenic fluids are known to sustain microbial consortia in shallow sediments, an in situ coupling of abiotic generation and microbial utilization has not been demonstrated. Here we show, using a combination of basin modelling, kinetic modelling, geomicrobiology and biogeochemistry, that microorganisms inhabit the active generation zone in the Nankai Trough, offshore Japan. Three sites from ODP Leg 190 have been evaluated, namely 1173, 1174 and 1177, drilled in nearly undeformed Quaternary and Tertiary sedimentary sequences seaward of the Nankai Trough itself. Paleotemperatures were reconstructed based on subsidence profiles, compaction modelling, present-day heat flow, downhole temperature measurements and organic maturity parameters. Today's heat flow distribution can be considered mainly conductive, and is extremely high in places, reaching 180 mW/m**2. The kinetic parameters describing total hydrocarbon generation, determined by laboratory pyrolysis experiments, were utilized by the model in order to predict the timing of generation in time and space. The model predicts that the onset of present day generation lies between 300 and 500 m below sea floor (5100-5300 m below mean sea level), depending on well location. In the case of Site 1174, 5-10% conversion has taken place by a present day temperature of ca. 85 °C. Predictions were largely validated by on-site hydrocarbon gas measurements. Viable organisms in the same depth range have been proven using 14C-radiolabelled substrates for methanogenesis, bacterial cell counts and intact phospholipids. Altogether, these results point to an overlap of abiotic thermal degradation reactions going on in the same part of the sedimentary column as where a deep biosphere exists. The organic matter preserved in Nankai Trough sediments is of the type that generates putative feedstocks for microbial activity, namely oxygenated compounds and hydrocarbons. Furthermore, the rates of thermal degradation calculated from the kinetic model closely resemble rates of respiration and electron donor consumption independently measured in other deep biosphere environments. We deduce that abiotically driven degradation reactions have provided substrates for microbial activity in deep sediments at this convergent continental margin.

Scaling up the production of biogenic magnetite nanoparticles

Although there are numerous examples of large-scale commercial microbial synthesis routes for organic bioproducts, few studies have addressed the obvious potential for microbial systems to produce inorganic functional biomaterials at scale. Here we address this by focusing on the production of nano-scale biomagnetite particles by the Fe(III)-reducing bacterium Geobacter sulfurreducens, which was scaled-up successfully from lab-scale to pilot plant-scale production, whilst maintaining the surface reactivity and magnetic properties which make this material well suited to commercial exploitation. At the largest scale tested, the bacterium was grown in a 50 L bioreactor, harvested and then inoculated into a buffer solution containing Fe(III)-oxyhydroxide and an electron donor and mediator, which promoted the formation of magnetite in under 24 hours. This procedure was capable of producing up to 120 g biomagnetite. The particle size distribution was maintained between 10 and 15 nm during scale-up of this second step from 10 ml to 10 L, with conserved magnetic properties and surface reactivity; the latter demonstrated by the reduction of Cr(VI). The process presented provides an environmentally benign route to magnetite production and serves as an alternative to harsher synthetic techniques, with the clear potential to be used to produce kg to tonne quantities.

Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Förster distance dependence and subpopulations

Photon bursts from single diffusing donor-acceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Förster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.