Biochemical analysis of mutations in P450 oxidoreductase

All microsomal P450s require POR (cytochrome P450 reductase) for catalytic activity. Most of the clinically used drugs are metabolized by a small number of P450s and polymorphisms in the cytochrome P450s are known to cause changes in drug metabolism. We have recently found a number of POR missense mutations in the patients with disordered steroidogenesis. Our initial report described five missense mutations (A284P, R454H, V489E, C566Y and V605F) identified in four patients. We built bacterial expression vectors for each POR variant, purified the membranes expressing normal or variant POR and characterized their activities with cytochrome c and P450c17 assays. We have recently completed an extensive study of the range of POR mutations and characterized the mutants/polymorphisms A112V, T139A, M260V, Y456H, A500V, G536R, L562P, R613X, V628I and F643del from sequencing of patient DNA. We also studied POR variants Y179D, P225L, R313W, G410S and G501R that were available in databases or the published literature. We analysed the mutations with a three-dimensional model of human POR that was based on an essentially similar rat POR with known crystal structure. The missense mutations found in patients with disordered steroidogenesis mapped to functionally important domains of POR and the apparent polymorphisms mapped to less crucial regions. Since a variation in POR can alter the activity of all microsomal P450s, it can also affect the drug metabolism even with a normal P450. Understanding the genetic and biochemical basis of POR-mediated drug metabolism will provide valuable information about possible differences in P450-mediated reactions among the individuals carrying a variant or polymorphic form of POR.

Monitoring pollution in river Mure , Romania, Part III: biochemical effect markers in fish and integrative reflection

Along a downstream stretch of River Mure , Romania, adult males of two feral fish species, European chub (Leuciscus cephalus) and sneep (Chondrostoma nasus) were sampled at four sites with different levels of contamination. Fish were analysed for the biochemical markers hsp70 (in liver and gills) and hepatic EROD activity, as well as several biometrical parameters (age, length, wet weight, condition factor). None of the biochemical markers correlated with any biometrical parameter, thus biomarker reactions were related to site-specific criteria. While the hepatic hsp70 level did not differ among the sites, significant elevation of the hsp70 level in the gills revealed proteotoxic damage in chub at the most upstream site, where we recorded the highest heavy metal contamination of the investigated stretch, and in both chub and sneep at the site right downstream of the city of Arad. In both species, significantly elevated hepatic EROD activity downstream of Arad indicated that fish from these sites are also exposed to organic chemicals. The results were indicative of impaired fish health at least at three of the four investigated sites. The approach to relate biomarker responses to analytical data on pollution was shown to fit well the recent EU demands on further enhanced efforts in the monitoring of Romanian water quality.

Deoxynucleoside triphosphates bearing histamine, carboxylic acid, and hydroxyl residues--synthesis and biochemical characterization.

Modified nucleoside triphosphates (dA(Hs)TP, dU(POH)TP, and dC(Val)TP) bearing imidazole, hydroxyl, and carboxylic acid residues connected to the purine and pyrimidine bases through alkyne linkers were prepared. These modified dN*TPs were excellent substrates for various DNA polymerases in primer extension reactions. Moreover, the combined use of terminal deoxynucleotidyl transferase (TdT) and the modified dNTPs led to efficient tailing reactions that rival those of natural counterparts. Finally, the triphosphates were tolerated by polymerases under PCR conditions, and the ensuing modified oligonucleotides served as templates for the regeneration of unmodified DNA. Thus, these modified dN*TPs are fully compatible with in vitro selection methods and can be used to develop artificial peptidases based on DNA.

Möller-Barlow disease in archaeology: Preliminary study of biochemical detection

Human bone is the most direct source for reconstructing health and living conditions of ancient populations. However, many diseases remain undetected in palaeopathology. Möller-Barlow disease (scurvy) is a historically well-documented metabolic disease and must have been common in clinical and sub-clinical severity. Due to long incubation periods and the subtle nature of bone changes osteological evidence is relatively rare (Brickley & Ives 2008). Möller-Barlow disease is caused by deficiency of dietary vitamin C (ascorbic acid) and evokes symptoms like fatigue, haemorrhage, inflammations, delayed wound healing and pain. Vitamin C is a cofactor for the hydroxylation of the amino acids proline and lysine which are essential for the production of intact connective tissue by cross-linking the propeptides in collagen. In a preliminary study we tested the detectability of Möller-Barlow disease by analysis of relative quantitative variability of hydroxylated amino acids in collagen (Pendery & Koon 2013). Samples (N=9) were taken from children with (n=3, cranium, femur, tibia) and without (n=4, cranium, femur, tibia) apparent bone reactions indicative of Möller-Barlow disease, as well as from adults with lethal traumata (n=2; negative controls). The skeletal remains originated from two early medieval cemeteries from Switzerland. Gas chromatographic (GC) analysis revealed minor differences between the samples. So far children with no pathologic alterations had fairly same values as negative controls while children with bone reactions paradoxically exhibited even slightly higher values of hydroxyproline and hydroxylysine. Future research demands for larger sample size and has to discuss sampling strategies. Beside possible misdiagnosis of Möller-Barlow disease it is arguable if only the newly built bone should be analysed even though this could lead to problems related to small sample quantity. It also remains to be seen to which extent varying turnover rates of different skeletal elements, especially in children, must be taken into account.

TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei.

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.

Proceedings of the Seventeenth Annual Biochemical Engineering Symposium

This is the seventeenth of a series of symposia devoted to talks by students about their biochemical engineering research. The first, third, fifth, ninth, twelfth, and sixteenth were at Kansas State University, the second and fourth were at the University of Nebraska-Lincoln, the sixth was in Kansas City and was hosted by Iowa State University, the seventh, tenth, thirteenth, and seventeenth were at Iowa State University, the eighth and fourteenth were at the University of Missouri–Columbia, and the eleventh and fifteenth were at Colorado State University. Next year's symposium will be at the University of Colorado. Symposium proceedings are edited by faculty of the host institution. Because final publication usually takes place elsewhere, papers here are brief, and often cover work in progress. ContentsThe Effect of Polymer Dosage Conditions on the Properties of ProteinPolyelectrolyte Precipitates, K. H. Clark and C. E. Glatz, Iowa State University An Immobilized Enzyme Reactor/Separator for the Hydrolysis of Casein by Subtilisin Carlsberg, A. J. Bream, R. A. Yoshisato, and G. R. Carmichael, University of Iowa Cell Density Measurements in Hollow Fiber Bioreactors, Thomas Blute, Colorado State University The Hydrodynamics in an Air-Lift Reactor, Peter Sohn, George Y. Preckshot, and Rakesh K. Bajpai, University of Missouri–Columbia Local Liquid Velocity Measurements in a Split Cylinder Airlift Column, G. Travis Jones, Kansas State University Fluidized Bed Solid Substrate Trichoderma reesei Fermentation, S. Adisasmito, H. N. Karim, and R. P. Tengerdy, Colorado State University The Effect of 2,4-D Concentration on the Growth of Streptanthus tortuosis Cells in Shake Flask and Air-Lift Permenter Culture, I. C. Kong, R. D. Sjolund, and R. A. Yoshisato, University of Iowa Protein Engineering of Aspergillus niger Glucoamylase, Michael R. Sierks, Iowa State University Structured Kinetic Modeling of Hybidoma Growth and Monoclonal Antibody Production in Suspension Cultures, Brian C. Batt and Dhinakar S. Kampala, University of Colorado Modelling and Control of a Zymomonas mobilis Fermentation, John F. Kramer, M. N. Karim, and J. Linden, Colorado State University Modeling of Brettanomyces clausenii Fermentation on Mixtures of Glucose and Cellobiose, Max T. Bynum and Dhinakar S. Kampala, University of Colorado, Karel Grohmann and Charles E. Yyman, Solar Energy Research Institute Master Equation Modeling and Monte Carlo Simulation of Predator-Prey Interactions, R. 0. Fox, Y. Y. Huang, and L. T. Fan, Kansas State University Kinetics and Equilibria of Condensation Reactions Between Two Different Monosaccharides Catalyzed by Aspergillus niger Glucoamylase, Sabine Pestlin, Iowa State University Biodegradation of Metalworking Fluids, S. M. Lee, Ayush Gupta, L. E. Erickson, and L. T. Fan, Kansas State University Redox Potential, Toxicity and Oscillations in Solvent Fermentations, Kim Joong, Rakesh Bajpai, and Eugene L. Iannotti, University of Missouri–Columbia Using Structured Kinetic Models for Analyzing Instability in Recombinant Bacterial Cultures, William E. Bentley and Dhinakar S. Kompala, University of Colorado

Biochemical evolution II: Origin of life in tubular microstructures on weathered feldspar surfaces

Mineral surfaces were important during the emergence of life on Earth because the assembly of the necessary complex biomolecules by random collisions in dilute aqueous solutions is implausible. Most silicate mineral surfaces are hydrophilic and organophobic and unsuitable for catalytic reactions, but some silica-rich surfaces of partly dealuminated feldspars and zeolites are organophilic and potentially catalytic. Weathered alkali feldspar crystals from granitic rocks at Shap, north west England, contain abundant tubular etch pits, typically 0.4–0.6 μm wide, forming an orthogonal honeycomb network in a surface zone 50 μm thick, with 2–3 × 106 intersections per mm2 of crystal surface. Surviving metamorphic rocks demonstrate that granites and acidic surface water were present on the Earth’s surface by ∼3.8 Ga. By analogy with Shap granite, honeycombed feldspar has considerable potential as a natural catalytic surface for the start of biochemical evolution. Biomolecules should have become available by catalysis of amino acids, etc. The honeycomb would have provided access to various mineral inclusions in the feldspar, particularly apatite and oxides, which contain phosphorus and transition metals necessary for energetic life. The organized environment would have protected complex molecules from dispersion into dilute solutions, from hydrolysis, and from UV radiation. Sub-micrometer tubes in the honeycomb might have acted as rudimentary cell walls for proto-organisms, which ultimately evolved a lipid lid giving further shelter from the hostile outside environment. A lid would finally have become a complete cell wall permitting detachment and flotation in primordial “soup.” Etch features on weathered alkali feldspar from Shap match the shape of overlying soil bacteria.

Progress toward chemical accuracy in the computer simulation of condensed phase reactions.

We describe a procedure for the generation of chemically accurate computer-simulation models to study chemical reactions in the condensed phase. The process involves (i) the use of a coupled semiempirical quantum and classical molecular mechanics method to represent solutes and solvent, respectively; (ii) the optimization of semiempirical quantum mechanics (QM) parameters to produce a computationally efficient and chemically accurate QM model; (iii) the calibration of a quantum/classical microsolvation model using ab initio quantum theory; and (iv) the use of statistical mechanical principles and methods to simulate, on massively parallel computers, the thermodynamic properties of chemical reactions in aqueous solution. The utility of this process is demonstrated by the calculation of the enthalpy of reaction in vacuum and free energy change in aqueous solution for a proton transfer involving methanol, methoxide, imidazole, and imidazolium, which are functional groups involved with proton transfers in many biochemical systems. An optimized semiempirical QM model is produced, which results in the calculation of heats of formation of the above chemical species to within 1.0 kcal/mol (1 kcal = 4.18 kJ) of experimental values. The use of the calibrated QM and microsolvation QM/MM (molecular mechanics) models for the simulation of a proton transfer in aqueous solution gives a calculated free energy that is within 1.0 kcal/mol (12.2 calculated vs. 12.8 experimental) of a value estimated from experimental pKa values of the reacting species.

Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

Biochemical characterization and DNA binding properties of the MocR family member GabR, a PLP-dependent transcription factor

GabR è un fattore di trascrizione chimerico appartenente alla famiglia dei MocR/GabR, costituito da un dominio N-terminale elica-giro-elica di legame al DNA e un dominio effettore e/o di oligomerizzazione al C-terminale. I due domini sono connessi da un linker flessibile di 29 aminoacidi. Il dominio C-terminale è strutturalmente omologo agli enzimi aminotransferasici fold-type I, i quali, utilizzando il piridossal-5’-fosfato (PLP) come cofattore, sono direttamente coinvolti nel metabolismo degli aminoacidi. L’interazione contemporanea di PLP e acido γ-aminobutirrico (GABA) a GabR fa sì che questa promuova la trascrizione di due geni, gabT e gabD, implicati nel metabolismo del GABA. GabR cristallizza come un omodimero con una configurazione testa-coda. Il legame con la regione promotrice gabTD avviene attraverso il riconoscimento specifico di due sequenze dirette e ripetute (ATACCA), separate da uno spacer di 34 bp. In questo studio sono state indagate le proprietà biochimiche, strutturali e di legame al DNA della proteina GabR di Bacillus subtilis. L’analisi spettroscopica dimostra che GabR interagisce con il PLP formando l’aldimina interna, mentre in presenza di GABA si ottiene l’aldimina esterna. L’interazione fra il promotore gabTD e le forme holo e apo di GabR è stata monitorata mediante Microscopia a Forza atomica (AFM). In queste due condizioni di legame è stata stimata una Kd di circa 40 ηM. La presenza di GABA invece, determinava un incremento di circa due volte della Kd, variazioni strutturali nei complessi GabR-DNA e una riduzione del compattamento del DNA alla proteina, indipendentemente dalla sequenza del promotore in esame. Al fine di valutare il ruolo delle caratteristiche topologiche del promotore, sono state inserite cinque e dieci bp all’interno della regione spacer che separa le due sequenze ripetute dirette riconosciute da GabR. I significativi cambiamenti topologici riscontrati nel frammento aggiunto di cinque bp si riflettono anche sulla forte riduzione dell’affinità di legame verso la proteina. Al contrario, l’inserzione di 10 bp provoca solamente l’allontanamento delle sequenze ripetute dirette. L’assenza quindi di cambiamenti significativi nella topologia di questo promotore fa sì che l’affinità di legame per GabR rimanga pressoché inalterata rispetto al promotore non mutato. L’analisi del potenziale elettrostatico superficiale di GabR mostra la presenza di una fascia carica positivamente che si estende lungo un’intera faccia della proteina. Per verificare l’importanza di questa caratteristica di GabR nel meccanismo di interazione al DNA, sono stati preparati ed indagati i mutanti R129Q e K362-366Q, in cui la carica positiva superficiale risultava indebolita. L’affinità di legame dei mutanti di GabR per il DNA era inferiore rispetto alla proteina non mutata, in particolar modo nel mutante K362-366Q. Le evidenze acquisite suggeriscono che la curvatura intrinseca del promotore ed il corretto orientamento delle sequenze sulla doppia elica, più della distanza che le separa, siano critici per sostenere l’interazione con GabR. Oltre a questo, la superficie positiva di GabR è richiesta per accomodare la curvatura del DNA sul corpo della proteina. Alla luce di questo, l’interazione GabR-gabTD è un esempio di come il riconoscimento specifico di sequenze, la topologia del DNA e le caratteristiche strutturali della proteina siano contemporaneamente necessarie per sostenere un’interazione proteina-DNA stabile.

A gradient descent algorithm for minimizing amino acid coupling reactions when synthesizing cyclic-peptide libraries

Combinatorial chemistry has become an invaluable tool in medicinal chemistry for the identification of new drug leads. For example, libraries of predetermined sequences and head-to-tail cyclized peptides are routinely synthesized in our laboratory using the IRORI approach. Such libraries are used as molecular toolkits that enable the development of pharmacophores that define activity and specificity at receptor targets. These libraries can be quite large and difficult to handle, due to physical and chemical constraints imposed by their size. Therefore, smaller sub-libraries are often targeted for synthesis. The number of coupling reactions required can be greatly reduced if the peptides having common amino acids are grouped into the same sub-library (batching). This paper describes a schedule optimizer to minimize the number of coupling reactions by rotating and aligning sequences while simultaneously batching. The gradient descent method thereby reduces the number of coupling reactions required for synthesizing cyclic peptide libraries. We show that the algorithm results in a 75% reduction in the number of coupling reactions for a typical cyclic peptide library.

Immunological and biochemical techniques in the analysis of tear proteins

This study is concerned with the analysis of tear proteins, paying particular attention to the state of the tears (e.g. non-stimulated, reflex, closed), created during sampling, and to assess their interactions with hydrogel contact lenses. The work has involved the use of a variety of biochemical and immunological analytical techniques for the measurement of proteins, (a), in tears, (b), on the contact lens, and (c), in the eluate of extracted lenses. Although a diverse range of tear components may contribute to contact lens spoilation, proteins were of particular interest in this study because of their theoretical potential for producing immunological reactions. Although normal host proteins in their natural state are generally not treated as dangerous or non-self, those which undergo denaturation or suffer a conformational change may provoke an excessive and unnecessary immune response. A novel on-lens cell based assay has been developed and exploited in order to study the role of the ubiquitous cell adhesion glycoprotein, vitronectin, in tears and contact lens wear under various parameters. Vitronectin, whose levels are known to increase in the closed eye environment and shown here to increase during contact lens wear, is an important immunoregulatory protein and may be a prominent marker of inflammatory activity. Immunodiffusion assays were developed and optimised for use in tear analysis, and in a series of subsequent studies used for example in the measurement of albumin, lactoferrin, IgA and IgG. The immunodiffusion assays were then applied in the estimation of the closed eye environment; an environment which has been described as sustaining a state of sub-clinical inflammation. The role and presence of a lesser understood and investigated protein, kininogen, was also estimated, in particular, in relation to contact lens wear. Difficulties arise when attempting to extract proteins from the contact lens in order to examine the individual nature of the proteins involved. These problems were partly alleviated with the use of the on-lens cell assay and a UV spectrophotometry assay, which can analyse the lens surface and bulk respectively, the latter yielding only total protein values. Various lens extraction methods were investigated to remove protein from the lens and the most efficient was employed in the analysis of lens extracts. Counter immunoelectrophoresis, an immunodiffusion assay, was then applied to the analysis of albumin, lactoferrin, IgA and IgG in the resultant eluates.

Stochastic Modeling of Reversible Biochemical Reaction-Diffusion Systems and High-Resolution Shock-Capturing Methods for Fluid Interfaces

Thesis (Ph.D.)--University of Washington, 2016-08

Microcantilever sensors for biochemical detection and analysis

Biochemical agents, including bacteria and toxins, are potentially dangerous and responsible for a wide variety of diseases. Reliable detection and characterization of small samples is necessary in order to reduce and eliminate their harmful consequences. Microcantilever sensors offer a potential alternative to the state of the art due to their small size, fast response time, and the ability to operate in air and liquid environments. At present, there are several technology limitations that inhibit application of microcantilever to biochemical detection and analysis, including difficulties in conducting temperature-sensitive experiments, material inadequacy resulting in insufficient cell capture, and poor selectivity of multiple analytes. This work aims to address several of these issues by introducing microcantilevers having integrated thermal functionality and by introducing nanocrystalline diamond as new material for microcantilevers. Microcantilevers are designed, fabricated, characterized, and used for capture and detection of cells and bacteria. The first microcantilever type described in this work is a silicon cantilever having highly uniform in-plane temperature distribution. The goal is to have 100 μm square uniformly heated area that can be used for thermal characterization of films as well as to conduct chemical reactions with small amounts of material. Fabricated cantilevers can reach above 300C while maintaining temperature uniformity of 2−4%. This is an improvement of over one order of magnitude over currently available cantilevers. The second microcantilever type is a doped single crystal silicon cantilever having a thin coating of ultrananocrystalline diamond (UNCD). The primary application of such a device is in biological testing, where diamond acts as a stable, electrically isolated reaction surface while silicon layer provides controlled heating with minimum variations in temperature. This work shows that composite cantilevers of this kind are an effective platform for temperature-sensitive biological experiments, such as heat lysing and polymerase chain reaction. The rapid heat-transfer of Si-UNCD cantilever compromised the membrane of NIH 3T3 fibroblast and lysed the cell nucleus within 30 seconds. Bacteria cells, Listeria monocytogenes V7, were shown to be captured with biotinylated heat-shock protein on UNCD surface and 90% of all viable cells exhibit membrane porosity due to high heat in 15 seconds. Lastly, a sensor made solely from UNCD diamond is fabricated with the intention of being used to detect the presence of biological species by means of an integrated piezoresistor or through frequency change monitoring. Since UNCD diamond has not been previously used in piezoresistive applications, temperature-denpendent piezoresistive coefficients and gage factors are determined first. The doped UNCD exhibits a significant piezoresistive effect with gauge factor of 7.53±0.32 and a piezoresistive coefficient of 8.12×10^−12 Pa^−1 at room temperature. The piezoresistive properties of UNCD are constant over the temperature range of 25−200C. 300 μm long cantilevers have the highest sensitivity of 0.186 m-Ohm/Ohm per μm of cantilever end deflection, which is approximately half that of similarly sized silicon cantilevers. UNCD cantilever arrays were fabricated consisting of four sixteen-cantilever arrays of length 20–90 μm in addition to an eight-cantilever array of length 120 μm. Laser doppler vibrometry (LDV) measured the cantilever resonant frequency, which ranged as 218 kHz−5.14 MHz in air and 73 kHz−3.68 MHz in water. The quality factor of the cantilever was 47−151 in air and 18−45 in water. The ability to measure frequencies of the cantilever arrays opens the possibility for detection of individual bacteria by monitoring frequency shift after cell capture.

Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM and HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.