A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.     

Characterisation of the flavin adenine dinucleotide binding region of Myxococcus xanthus protoporphyrinogen oxidase

Protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the haem biosynthetic pathway catalysers the six electron oxidation of protoporphyrinogen-IX to protoporphyrin-IX, in the presence of flavin adenine dinucleotide (FAD) and oxygen. In humans, partial defects in PPOX result in variegate porphyria. In this study, the FAD binding region in Myxococcus xanthus PPOX was analysed by engineering and characterising a selection of mutant proteins. Amino acid residues which interact with FAD via their side chains were selected for study. Mutants were characterised and compared with wild type protein. Characterisation included FAD quantitation, analysis of FAD spectra and kinetic assay. Results revealed that Serine 20 mutants could still bind FAD, but polarity in this position is favourable, yet not essential for the integrity of FAD binding. Study of Glutamate 39 mutants suggest that a negative charge at position 39 is clearly favoured for interaction with the ribose ring of FAD, as all non-conservative replacements could not bind sufficient FAD. Asparagine 441 appears not to be directly involved in FAD binding but rather in stabilizing the FAD, and polarity in this position appears important. Tryptophan 408 may play a role in orientating or stabilizing the bound substrate during catalysis, and a non-polar (or slightly polar) residue is favoured at this position; however, aromaticity in this position appears not to be critical. Overall this study sheds further light on how M. xanthus PPOX interacts with FAD.     

Mitochondrial targeting of human protoporphyrinogen oxidase

Variegate porphyria is an autosomal dominant disorder of heme metabolism resulting from a deficiency in protoporphyrinogen oxidase, an enzyme located on the inner mitochondrial membrane. This study examined the effect of three South African VP-causing mutations (H20P, R59W, R168C) on mitochondrial targeting. Only H20P did not target, and of eight protoporphyrinogen oxidase-GFP chimeric fusion proteins created, N-terminal residues 1-17 were found to be the minimal protoporphyrinogen oxidase sequence required for efficient mitochondrial targeting. Removal of this N-terminal sequence displayed mitochondrial localization, suggesting internal mitochondrial targeting signals. In addition, six constructs were engineered to assess the effect of charge and helicity on mitochondrial targeting of the protein. Of those engineered, only the PPOX20/H20P-GFP construct abolished mitochondrial targeting, presumably through disruption of the protoporphyrinogen oxidase alpha-helix. Based on our results we propose a mechanism for protoporphyrinogen oxidase targeting to the mitochondrion.     

Design and synthesis of 1-(benzothiazol-5-yl)-1H-1,2,4-triazol-5-ones as protoporphyrinogen oxidase inhibitors

Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is the action target for several structurally diverse herbicides. A series of novel 4-(difluoromethyl)-1-(6-halo-2-substituted-benzothiazol-5-yl)-3-methyl-1H-1,2,4-triazol-5(4H)-ones 2a–z were designed and synthesized via the ring-closure of two ortho-substituents. The in vitro bioassay results indicated that the 26 newly synthesized compounds exhibited good PPO inhibition effects with K_i values ranging from 0.06 to 17.79 μM. Compound 2e, ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzo-thiazol-2-yl]thio}acetate, was the most potent inhibitor with K_i value of 0.06 μM against mtPPO, comparable to (K_i = 0.03 μM) sulfentrazone. Further green house assays showed that compound 2f (K_i = 0.24 μM, mtPPO), ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzothiazol-2-yl]thio}propanoate, showed the most promising post-emergence herbicidal activity with broad spectrum even at concentrations as low as 37.5 g ai/ha. Soybean exhibited tolerance to compound 2f at the dosages of 150 g ai/ha, whereas they are susceptible to sulfentrazone even at 75 g ai/ha. Thus, compound 2f might be a potential candidate as a new herbicide for soybean fields.     

Rapid screening of monoamine oxidase B inhibitors in natural extracts by capillary electrophoresis after enzymatic reaction at capillary inlet

A facile capillary electrophoresis (CE) method was developed for the screening of monoamine oxidase B (MAO-B) inhibitors in natural extracts. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds, and detected by their UV absorbance at 280 nm. Conditions for the separation of substrates, products and enzyme were optimized. The optimal buffer composition was 50 mM N-2-hydroxyethyl-piperazine-N′-2-ethane sulphonic acid (HEPES) solution containing 10 mM SDS (pH = 7.4). Under the optimal condition, the baseline separation of substrates, products and enzyme was achieved within 2 min. The present method was used to determine MAO-B kinetic constants, K_i, K_m and IC₅₀ based on quantitative of the substrate peak area compared with the reference electropherogram obtained from without the inhibitor. A validation study shows good reproducibility for both migration time (RSD = 1.8%) and peak area (RSD = 3.9%). Finally, the screening of 16 natural extracts was performed, and 2 natural extracts from Fructus crataegi and Radix polygoni multiflori were identified to be positive for MAO-B inhibition.     

Expression of recombinant protoporphyrinogen oxidase influences growth and morphological characteristics in transgenic rice

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase (Protox), the last shared enzyme of the porphyrin pathway, in the cytoplasm (C89) or the plastids (P72) were compared with wild-type rice plants in their growth characteristics. Production of tiller buds 18 d after seeding was more profuse in transgenic plants than in wild-type plants, especially in plastid-targeted plants. Transgenic plants had 12–27% increase in tiller number and 17–33% increase in above-ground biomass compared with wild-type plants 4 and 8 weeks after transplanting of 2-week-old rice seedlings, demonstrating that tiller production and above-ground biomass correlate with each other. Cytoplasm-expressed and plastid-targeted transgenic plants also had a distinct phenotypic characteristic of narrower and more horizontal leaves than wild-type plants. Phenotypic and anatomical characteristics of the transgenic plants were clearly different from wild-type plants, indicating that regulation of porphyrin biosynthesis by expression of B. subtilis Protox in rice influences morphological characteristics of plant growth as well as biomass.     

A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

A continuous spectrofluorimetric assay for protoporphyrinogen oxidase (PPO, EC 1.3.3.4) activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless nonfluorescent compound. The evolution of the fluorescent tetrapyrrole product, protoporphyrin IX, was detected using a fluorescence plate reader. The apparent Km (Kapp) values for protoporphyrinogen IX were measured as 3.8+/-0.3, 3.6+/-0.5, and 1.0+/-0.1 microM for the enzymes from human, Myxococcus xanthus, and Aquifex aeolicus, respectively. The Ki for acifluorfen, a diphenylether herbicide, was measured as 0.53 microM for the human enzyme. Also, the specific activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h-1/mg mitochondria, demonstrating that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as low as 0.5 nM min-1, representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this continuous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will be of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples.     

1,4-Benzoquinone-based electrophoretic assay for glucose oxidase

The communication demonstrates feasibility of an enzyme microassay for glucose oxidase with 1,4-benzoquinone as an acceptor of electrons. The protocol uses the plug-plug mode of electrophoretically mediated microanalysis, with nanolitre injected volumes of enzyme and reactant solutions. The reactant and product, 1,4-benzoquinone and hydroquinone, are separated during the assay by differential binding to sulfated-beta-cyclodextrin used as additive to the phosphate buffer (pH 7) and monitored at selected wavelengths in their UV spectra. The assay covers glucose oxidase concentration from 0.01 to 0.1mgml(-1). Due to the strong UV absorbance of the both reactant and product, there is no need for use of a second enzyme (peroxidase) in the present assay.     

Analysis of single nucleotide incorporation reactions by capillary electrophoresis

Single nucleotide incorporation assays have been used to probe the kinetic parameters of many DNA and RNA polymerases. Traditionally, oligonucleotide primers are 5'-(32)P labeled using T4 kinase and annealed to a complementary template with a 5' overhang. To quantify the reaction kinetics, the products of the primer extension reactions are usually separated using denaturing polyacrylamide gel electrophoresis and quantified using a phosphorimager or other method to measure radioactivity. We have developed a method using a 5' fluorescently labeled oligonucleotide to examine the kinetics of single nucleotide incorporation catalyzed by recombinant human mitochondrial polymerase gamma (Pol gamma) holoenzyme. Using laser-induced fluorescence detection in the P/ACE MDQ instrument, primers 5' labeled with fluorescent probes such as 6-carboxyfluorescein can be rapidly separated and quantified. However, we also show that only select probes can be used, presumably due to unfavorable interactions between Pol gamma and certain 5' labels.     

Functional analysis of the hemK gene product involvement in protoporphyrinogen oxidase activity in yeast

The Escherichia coli hemK gene has been described as being involved in protoporphyrinogen oxidase activity; however, there is no biochemical evidence for this. In the context of characterizing the mechanisms of protoporphyrinogen oxidation in the yeast Saccharomyces cerevisiae, we investigated the yeast homolog of HemK, which is encoded by the ORF YNL063w, to find out whether it has any protoporphyrinogen oxidase activity and/or whether it modulates protoporphyrinogen oxidase activity. Phenotype analysis and enzyme activity measurements indicated that the yeast HemK homolog is not involved in protoporphyrinogen oxidase activity. Complementation assays in which the yeast HemK homolog is overproduced do not restore wild-type phenotypes in a yeast strain with deficient protoporphyrinogen oxidase activity. Protein sequence analysis of HemK-related proteins revealed consensus motif for S-adenosyl-methionine-dependent methyltransferase.     

A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: implications for substrate specificity

The potent botulinum neurotoxin inhibits neurotransmitter release at cholinergic nerve terminals, causing a descending flaccid paralysis characteristic of the disease botulism. The currently expanding medical use of the neurotoxin to treat several disorders, as well as the potential misuse of the neurotoxin as an agent in biowarfare, has made understanding of the nature of the toxin's catalytic activity and development of inhibitors critical. To study the catalytic activity of botulinum neurotoxin more thoroughly and characterize potential inhibitors, we have developed a capillary electrophoresis method to measure catalytic activity of different serotypes of botulinum neurotoxin using peptides derived from the native substrates. This assay requires only a minute amount of sample (25 nl), is relatively rapid (15 min/sample), and allows the determination of enzyme kinetic constants for a more sophisticated characterization of inhibitors and neurotoxin catalytic activity. Using this method, we can measure activity of five of the seven serotypes of botulinum neurotoxin (A, B, E, F, and G) with two peptide substrates. Botulinum neurotoxin serotypes C and D did not cleave our peptides, lending insight into potential substrate requirements among the serotypes.     

High-resolution glycoprotein analysis using capillary electrophoresis

The high-resolution separation achievable with capillary electrophoresis has been applied successfully to the analysis of glycoproteins. Inherent in the implementation of this technology for glycoprotein analysis is the use of specific buffer additives. Bifunctional cationic reagents, such as simple alkyl chains bearing terminal amino or quaternary ammonium groups, have been particularly useful for the analysis of ovalbumin, an excellent model glycoprotein. Although dynamic coating of the capillary wall and the subsequent decrease in protein-wall interactions is known to be key in the effectiveness of these additives, much remains to be learned regarding the mechanism through which they function.     

Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane-embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenyl-pyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD-binding domain of the p-hydroxybenzoate-hydrolase fold and a substrate-binding domain that enclose a narrow active site cavity beneath the FAD and an alpha-helical membrane-binding domain. The active site architecture suggests a specific substrate-binding mode compatible with the unusual six-electron oxidation. The membrane-binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO.     

Analysis of glycated hemoglobin A1c by capillary electrophoresis and capillary isoelectric focusing

Two capillary electrophoretic methods were developed and evaluated for measurement of glycated hemoglobin A1c (HbA1c). First, a capillary electrophoresis analysis is performed with a sodium tetraborate buffer (pH 9.3) as background electrolyte in a neutrally coated capillary. HbA1c is separated from HbA0 due to specific interactions of borate anions with the cis–diol pattern in the saccharide moiety of glycohemoglobin. Second, a capillary isoelectric focusing method, which exploits a difference in pI values of HbA0 and HbA1c, is performed with Servalyt pH 6–8 or alternatively with Biolyte pH 6–8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by preparative fractionation of Servalyt pH 4–9 carrier ampholytes. Both methods reflect recent developments in the methodology of capillary electrophoresis. They allow quantifying HbA1c in generic capillary electrophoresis analyzer with specificity that is consistent with previously reported electrophoretic assays in slab gels and capillaries.     

On-line melting of double-stranded DNA for analysis of single-stranded DNA using capillary electrophoresis

Capillary electrophoresis (CE) is a convenient, fast and non-radioactive method with possibilities for automatization. To analyse single-stranded DNA molecules in a more automated way, we developed a heating device to melt double-stranded DNA fragments in the capillary during electrophoresis. In this study we used this device to obtain single-stranded DNA, necessary for the detection of point mutations in DNA using the single-strand conformation polymorphism technique. Results show that double-stranded DNA molecules can be melted on-line into single-stranded DNA molecules, although not for 100%. In an attempt to find universal electrophoretic conditions for the analysis of single-stranded DNA, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-stranded DNA peaks. We demonstrate that this heating device is a technical adjustment of CE which contributes to more automated analyses of DNA fragments.     

A recommended workflow for DNase I footprinting using a capillary electrophoresis genetic analyzer

Fragment analysis was developed to determine the sizes of DNA fragments relative to size standards of known lengths using a capillary electrophoresis genetic analyzer. This approach has since been adapted for use in DNA footprinting. However, DNA footprinting requires accurate determination of both fragment length and intensity, imposing specific demands on the experimental design. Here we delineate essential considerations involved in optimizing the fragment analysis workflow for use in DNase I footprinting to ensure that changes in DNase I cleavage patterns may be reliably identified.     

A gold nanoparticle-mediated enzyme bioreactor for inhibitor screening by capillary electrophoresis

A facile protocol to prepare highly effective and durable in-line enzyme bioreactors inside capillary electrophoresis (CE) columns was developed. To demonstrate the methodology, l-glutamic dehydrogenase (GLDH) was selected as the model enzyme. GLDH was first immobilized onto 38-nm-diameter gold nanoparticles (GNPs), and the functionalized GNPs were then assembled on the inner wall at the inlet end of the CE capillary treated with polyethyleneimine (PEI), producing an in-line GLDH bioreactor. Compared with a GLDH bioreactor prepared by immobilizing GLDH directly on PEI-treated capillary, the GNP-mediated bioreactor showed a higher enzymatic activity and a much better stability. The in-capillary enzyme bioreactor was proven to be very useful for screening of GLDH inhibitors deploying the GLDH-catalyzed α-ketoglutaric acid reaction. The screening assay was preliminarily validated by using a known GLDH inhibitor, namely perphenazine. A Z′ factor value of 0.95 (n = 10) was obtained, indicating that the screening results were highly reliable. Screening of GLDH inhibitors present in medicinal plant extracts by the proposed method was demonstrated. The inhibition percentages were found to be 53% for Radix scutellariae, 45% for Radix codonopsis, 37% for Radix paeoniae alba, and 0% for the other 22 extracts tested at a concentration of 0.6 mg extract/ml.     

A capillary electrophoresis-based enzyme assay for kinetics and inhibition studies of carbonic anhydrase

In the current study, capillary electrophoresis (CE)-based enzyme assay for characterization and inhibition study of bovine carbonic anhydrase II (bCA II) was developed. The developed method is the first CE assay for carbonic anhydrase (CA). The method was optimized in order to get short analysis time, minimal sample volume consumption, and high resolution of substrate and product. The CE conditions were optimized as follows: fused-silica capillary (30 cm effective length × 75 μm i.d.), pressure injection for 5 s, 20 mM sodium borate buffer (pH 9.0), constant voltage of 15 kV, constant capillary temperature of 25 °C, and detection at 260 nm. For precise measurements, uridine was used as an internal standard during optimization of the CE methods. The limits of detection and quantification for p-nitrophenyl acetate (p-NPA) were 3.01 and 9.12 μM, respectively, whereas for p-nitrophenolate they were 2.05 and 6.22 μM, respectively. The performance of the developed method was confirmed by determination of kinetic parameters (i.e., K_m and V_max of bCA for p-NPA); the inhibition constant (K_i) was determined for furosemide, a standard inhibitor of CA. The new method proved to be fast and efficient, and it can be used for the investigation of inhibitors of all isoforms of CAs.