Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: identification of putative substrate access regions

To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu³⁵⁹-Lys³⁷³), peptide II (Leu³⁰-Arg⁴⁸), peptide III (Phe¹²⁷-Arg¹⁴⁰), and peptide IV (Arg⁴³⁴-Arg⁴⁴³). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg¹³³, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.     

Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant

All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays a crucial role in their mechanism of action. Conversion of the proximal Cys436 to Ser in NH₂-truncated microsomal CYP2B4 (ΔCYP2B4) transforms the enzyme into a two-electron NADPH oxidase producing H₂O₂ without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542–553]. To examine the effects of this ligation change on the heme iron spin-state and coordination structure of ΔC436S CYP2B4, the magnetic circular dichroism and electronic absorption spectra of several oxidation/ligation states of the variant have been measured and compared with those of structurally defined heme complexes. The spectra of the substrate-free ferric mutant are indicative of a high-spin five-coordinate structure ligated by anionic serinate. The spectroscopic properties of the dithionite-reduced (deoxyferrous) protein are those of a five-coordinate (high-spin) state, and it is concluded that the proximal ligand has been protonated to yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes of the mutant with neutral sixth ligands (NO, CO, and O₂) examined are also likely ligated by neutral serine, as would be expected for ferric complexes with anionic sixth ligands such as the hydroperoxo-ferric catalytic intermediate. Ligation of the heme iron by neutral serine vs. deprotonated cysteine is likely the result of the large difference in their acidity. Thus, without the necessary proximal ligand push of the cysteinate, although the ΔC436S mutant can accept two electrons and two protons, it is unable to heterolytically cleave the O–O bond of the hydroperoxo-ferric species to generate Compound I and hydroxylate the substrate.     

Characterization of the DNA-binding domain and identification of the active site residue in the 'Gyr A' half of Leishmania donovani topoisomerase II

DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. To investigate the biochemical properties of the individual domains of Leishmania donovani topoisomerase II, four truncation mutants were generated. Deletion of 178 aminoacids from the C-terminus (core and LdΔC1058) had no apparent effect on the DNA-binding or cleavage activities of the enzymes. However, when 429 aminoacids from the N-terminus and 451 aminoacids from the C-terminus were removed (LdΔNΔC), the enzyme was no longer active. Moreover, the removal of 429 aminoacids from the N-terminus (LdΔNΔC, core and LdΔN429) render the mutant proteins incapable of performing ATP hydrolysis. The mutant proteins show cleavage activities at wide range of KCl concentrations (25–350 mM). In addition, the mutant proteins, excepting LdΔNΔC, can also act on kDNA and linearize the minicircles. Surprisingly, the mutant proteins fail to show the formation of the enhanced cleavable complex in the presence of etoposide. Our findings suggest that the conformation required for interaction with the drug is absent in the mutant proteins. Here, we have also identified Tyr⁷⁷⁵ through direct sequencing of the DNA linked peptide as the catalytic residue implicated in DNA-breakage and rejoining. Taken together, our results demonstrate that topoisomerase II are functionally and mechanistically conserved enzymes and the variations in activity seem to reflect functional optimization for its physiological role during parasite genome replication.     

The voltage-sensing domain of a hERG1 mutant is a cation-selective channel

A cationic leak current known as an “omega current” may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li⁺, K⁺, Cs⁺, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.     

The role of Arginine 38 in horseradish peroxidase enzyme revisited: A computational investigation

Molecular dynamics simulations on hydrogen peroxide complex with wild-type (WT) and Arg38Leu mutated (R38L) Horseradish Peroxidase (HRP) were carried out over nanoseconds timescale in water solution at 300 K. Comparison of the results provides interesting insights about the role of highly conserved Arg38 and His42 residues in the chemical features of HRP, underlying its biological activity which initiates with Compound0 (Cpd0). In the WT-HRP enzyme current molecular dynamics simulations show, for the first time, that Arg38 residue: i) prevents the entrance of water inside the reaction cavity, hence providing a hydrophobic reactive scenario, ii) it maintains the distance between His42 and heme–H₂O₂ complex suitable for the occurrence of proton transfer reaction leading, thereafter, to heme–H₂O₂ disruption according to Poulos-Kraut mechanism. On the other hand, R38L mutant can be considered as a “wet enzyme” where the presence of water solvent molecules in the heme reaction pocket, unfavoring the initial heme–H₂O₂ complex formation, decreases the catalytic efficiency in agreement with experimental kinetics measurements. Furthermore, we note that Arg38Leu mutation pushes the His42 residue far from the heme–H₂O₂ complex, making unlikely a direct proton transfer and suggesting that, in the mutant, a solvent water molecule could be involved in the first step of the Poulos-Kraut mechanism.     

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.     

Covalent heme binding to CYP4B1 via Glu310 and a carbocation porphyrin intermediate

Recently we found that CYP4B1, and several other members of the CYP4 family of enzymes, are covalently linked to their prosthetic heme group through an ester linkage. In the current study, we mutated a conserved CYP4 I-helix residue, E310 in rabbit CYP4B1, to glycine, alanine, and aspartate to examine the effect of these mutations on the extent of covalent heme binding and catalysis. All mutants expressed well in insect cells and were isolated as a mixture of monomeric and dimeric forms as determined by LC/ESI-MS of the intact proteins. Rates of metabolism decreased in the order E310 > A310 > G310 > D310, with the A310 and G310 mutants exhibiting alterations in regioselectivity for omega-1 and omega-2 hydroxylation of lauric acid, respectively. In marked contrast to the wild-type E310 enzyme, the G310, A310, and D310 mutants did not bind heme covalently. Uniquely, the acid-dissociable heme obtained from the D310 mutant contained an additional 16 amu relative to heme and exhibited the same chromatographic behavior as the monohydroxyheme species released upon base treatment of the covalently linked wild-type enzyme. Expression studies with H(2)(18)O demonstrated incorporation of the heavy isotope from the media into the monohydroxyheme isolated from the D310 mutant at a molar ratio of approximately 0.8:1. These data show (i) that E310 serves as the site of covalent attachment of heme to the protein backbone of rabbit CYP4B1; (ii) this I-helix glutamate residue influences substrate orientation in the active site of CYP4B1; and (iii) the mechanism of covalent heme attachment most likely involves a carbocation species located on the porphyrin.     

Assay of microsomal oxysterol 7α-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols

A method of assaying hepatic cytochrome P-450, oxysterol 7α-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27-³H]hydroxycholesterol as a substrate and hydroxypropyl-β-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7α,25-dihydroxycholesterol, into 3-oxo-7α,25-dihydroxy-4-cholesten by the activity of 3β-hydroxy-Δ⁵-C₂₇ steroid oxydoreductase, a microsomal NAD⁺ and NADP⁺ dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP⁺ into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7α-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (?33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (?30%) and also by the oxysterols 7α-hydroxycholesterol (?21%), 7β-hydroxycholesterol (?25%) and epicoprostanol (?20%), and by cyclosporin A (?26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols.     

A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum

In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn²⁺.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn²⁺.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.     

Studies on active site mutants of P. falciparum adenylosuccinate synthetase: Insights into enzyme catalysis and activation

Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg²⁺ to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coli AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K_m values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k_cat value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the biochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coli AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis.     

Time-dependent irreversible inhibition of bovine kidney alkaline phosphatase by oxidized adenosine. Use of this compound as a site-directed inhibitor for studying uncompetitive inhibition

The L/B/K type of mammalian alkaline phosphatase (ALP) is inhibited uncompetitively by nucleotides. A combination of adenosine and nicotinamide is more effective than either adenosine or nicotinamide alone, probably because a dinucleotide structure is necessary to trigger a conformational change accompanying binding of structures such as NADH. It has been suggested that a loop region containing residue 429 in the ALP polypeptide is important in the interaction of uncompetitive inhibitors with the enzyme. In the L/B/K isoenzyme, residue 429 is a histidine and is a potential target for modification. In an attempt to learn more about the molecular events accompanying inhibition of ALP by uncompetitive inhibitors, bovine kidney ALP was reacted with oxidized adenosine in the presence of nicotinamide to see if site-directed modification occurs. Kidney ALP was irreversibly inactivated by oxidized adenosine but the reaction was slow. The site modified is likely to be close to the region of binding. Sequence data for the kidney enzyme shows that in the region of residue 429 there are no residues except His⁴²⁹ itself that is likely to react with oxidized adenosine.     

Improving the affinity and activity of CYP101D2 for hydrophobic substrates

CYP101D2 is a cytochrome P450 monooxygenase from Novosphingobium aromaticivorans which is closely related to CYP101A1 (P450cam) from Pseudomonas putida. Both enzymes selectively hydroxylate camphor to 5-exo-hydroxycamphor, and the residues that line the active sites of both enzymes are similar including the pre-eminent Tyr96 residue. However, Met98 and Leu253 in CYP101D2 replace Phe98 and Val247 in CYP101A1, and camphor binding only results in a maximal change in the spin state to 40 % high-spin. Substitutions at Tyr96, Met98 and Leu253 in CYP101D2 reduced both the spin state shift on camphor binding and the camphor oxidation activity. The Tyr96Ala mutant increased the affinity of CYP101D2 for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane. The monooxygenase activity of the Tyr96Ala variant towards alkane substrates was also enhanced compared with the wild-type enzyme. The crystal structure of the substrate-free form of this variant shows the enzyme in an open conformation (PDB: 4DXY), similar to that observed with the wild-type enzyme (PDB: 3NV5), with the side chain of Ala96 pointing away from the heme. Despite this, the binding and activity data suggest that this residue plays an important role in substrate binding, evidencing that the enzyme probably undergoes catalysis in a more closed conformation, similar to those observed in the crystal structures of CYP101A1 (PDB: 2CPP) and CYP101D1 (PDB: 3LXI).     

Effect of Conformational Dynamics on Substrate Recognition and Specificity as Probed by the Introduction of a de Novo Disulfide Bond into Cytochrome P450 2B1

The conformational dynamics of cytochrome P450 2B1 (CYP2B1) were investigated through the introduction of a disulfide bond to link the I- and K-helices by generation of a double Cys variant, Y309C/S360C. The consequences of the disulfide bonding were examined both experimentally and in silico by molecular dynamics simulations. Under high hydrostatic pressures, the partial inactivation volume for the Y309C/S360C variant was determined to be −21 cm³mol⁻¹, which is more than twice as much as those of the wild type (WT) and single Cys variants (Y309C, S360C). This result indicates that the engineered disulfide bond has substantially reduced the protein plasticity of the Y309C/S360C variant. Under steady-state turnover conditions, the S360C variant catalyzed the N-demethylation of benzphetamine and O-deethylation of 7-ethoxy-trifluoromethylcoumarin as the WT did, whereas the Y309C variant retained only 39% of the N-demethylation activity and 66% of the O-deethylation activity compared with the WT. Interestingly, the Y309C/S360C variant restored the N-demethylation activity to the same level as that of the WT but decreased the O-deethylation activity to only 19% of the WT. Furthermore, the Y309C/S360C variant showed increased substrate specificity for testosterone over androstenedione. Molecular dynamics simulations revealed that the engineered disulfide bond altered substrate access channels. Taken together, these results suggest that protein dynamics play an important role in regulating substrate entry and recognition.Liver microsomal cytochromes P450 (CYP or P450)² metabolize a large number of clinically used drugs that have diverse steric and functional moieties. Despite low sequence homology among CYPs from different families, all P450s invariably contain a heme cofactor that is coordinated to a thiolate and catalyze the oxidative metabolism, mostly through hydroxylation, of substrates. However, production of reactive intermediates by P450s is often associated with drug toxicity and carcinogenesis, and inhibition or induction of a specific P450 isoform may lead to adverse drug-drug interactions (1). From a clinical and pharmacological perspective, it is important to understand the structure, function, and dynamics of P450s.Structural studies of P450s by x-ray crystallography in the past decade have provided us with a wealth of information regarding the structural organization, critical active site residues, and proton delivery pathways of P450s (2–4). In particular, these structural analyses have consistently shown that certain regions of the P450 structures such as the F/G and B/B′-C loops are extremely flexible and can undergo large conformational changes to accommodate substrates of various sizes, although the overall folding pattern of all P450s is conserved. For instance, an open conformation was observed in the ligand-free CYP2B4 crystal structure, whereas a closed conformation was reported for the CPI-bound CYP2B4 (3, 5). The open-to-closed conformational change involves large motions of the F- and G-helices and the F/G and B/B′-C loops. Based on comparisons of the crystal structures of CYP2B4 bound with inhibitors of different sizes, Zhao et al. (6) identified five plastic regions in P450s, including the B/B′-C loop (PR2) and F/G loop (PR4). Binding of ketoconazole or erythromycin to CYP3A4 led to a large increase in the active site volume (>80% increase) because of conformational changes primarily in the PR4, but interestingly the F- and G-helices moved in the opposite direction (7). These authors proposed that the extreme flexibility of CYP3A4 accounts for its promiscuity, as CYP3A4 metabolizes nearly ∼50% of all clinically used drugs. The complexity of the conformational flexibility and dynamics are also revealed in an MD simulation study of CYP3A4, 2C9 and 2A6 (8). Importantly, this molecular dynamics (MD) simulation study shows that the three-dimensional structure of P450s is more flexible in solution than was observed in the crystal structure.Despite intensive studies of the crystal structures of microsomal P450s, insights into the conformational dynamics of P450s in solution, particularly in relation to their functional importance, are lacking. A laser flash photolysis study of CO rebinding to CYP2E1 in solution revealed that the binding of substrates such as ethanol, pyrazole, and acetaminophen restricts the conformational flexibility of CYP2E1, as the kinetics for the rebinding of CO to ligand-bound CYP2E1 are significantly slower than those for the ligand-free CYP2E1 (9). A solution thermodynamics study of CYP2B4 supports the notion that CYP2B4 is remarkably flexible, as the entropy substantially decreases upon inhibitor binding resulting from reduction of the hydrophobic surface (10). In this study, a de novo disulfide bond is engineered into CYP2B1 and the consequences resulting from the disulfide bonding are examined both experimentally and in silico using MD simulations. To discern the effect of the de novo disulfide bond apart from the Cys mutagenesis, both the single and double Cys variants were characterized in detail. To our knowledge, this is the first report that investigates the consequences of limiting conformational dynamics in a P450 by incorporating a disulfide bond. Our results demonstrate that protein dynamics play an important role in regulating substrate entry/product egress channels and substrate recognition and provide insights that will be valuable for rational drug design and protein engineering.     

Functional complementation of dwf4 mutants of Arabidopsis by overexpression of CYP724A1

An essential step in the biosynthesis of bioactive brassinosteroids (BRs) in plants is the hydroxylation at C-22, a reaction catalyzed by P450 enzymes of the CYP90B and CYP724B subfamilies. Genes for both types of enzymes are present in many species, and in rice (Oryza sativa) and tomato (Solanum lycopersicum) both CYP90B and CYP724B enzymes contribute to C-22 hydroxylation. In Arabidopsis (Arabidopsis thaliana), C-22 hydroxylation of BRs is catalyzed by CYP90B1 (encoded by DWF4) and null dwf4 mutants show severe symptoms of BR-deficiency. CYP724A1 (At5g14400), an Arabidopsis gene of unknown function and limited expression, encodes a P450 sharing less than 55% sequence identity to CYP724B proteins. We used transgenic plants of the null mutants dwf4-102 and a novel allele, bashful (bsf), ectopically expressing the CYP724A1 gene to investigate the potential activity of CYP724A1 as a C-22 hydroxylase of BRs. Defects associated with BR deficiency were reversed and a normal growth habit restored in transgenic dwf4-102 and bsf plants overexpressing CYP724A1. The vegetative phase was prolonged and the transgenic plants were on average larger than wild type plants with respect to several morphometric parameters. Fertility was restored in the transgenic plants but individual siliques yielded fewer and heavier seeds than those of wild type plants. The implications of these findings with regard to the functions of CYP724A1 and the activity of its encoded enzyme are discussed.     

Voltage-insensitive Gating after Charge-neutralizing Mutations in the S4 Segment of Shaker Channels

Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from −160 to +80 mV with a constant opening probability (P _o). Although P _o is low (∼0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca²⁺ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb⁺ is substituted for K⁺ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive P _o seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.     

Identification and characterization of six cytochrome P450 genes belonging to CYP4 and CYP6 gene families in the silkworm,Bombyx mori

It was predicted that the genome of silkworm, Bombyx mori, has at least 79 P450 genes; however, P450 genes that are related to the catabolism of exogenous compounds were not reported. In this study we cloned two CYP4 (named CPY4M5 and CYP4M9) and four CYP6 (named CYP6AB5, CYP6AE9, CYP6AE22 and CYP6AU1) genes by using both bioinformatics and RT-PCR approaches. Sequence analysis showed that these genes contained conserved P450 gene sequence regions and one conserved intron. CYP4M5 and CYP4M9 genes were clustered together in a mode of “head-to-tail” possibly due to gene duplication. Blast analysis showed that these P450 genes shared significant similarity with CYP4 and CYP6 genes that are involved in the catabolism and detoxification of exogenous compounds in other insect species. RT-PCR results showed that these P450 genes were highly expressed in the midgut and fat body of B. mori. As the instar age increased, these P450 genes exhibit different expression patterns. When B. mori was exposed to 1.75 × 10^?5 % of cypermethrin, 3.5 × 10^?6 % of cypermethrin and 0.1 % of rutin, expression of CYP6AB5 was increased by 2.3-fold, 2.2-fold and 1.9-fold, respectively. Exposure of B. mori to 0.1 % quercetin does not change the expression of CYP6AB5. In contrast, expression of the other five P450 genes was inhibited after exposed to these compounds.     

Novel homozygous p.Y395X mutation in the CYP11B1 gene found in a Vietnamese patient with 11β-hydroxylase deficiency

Context

The deficiency of steroid 11β-hydroxylase is caused by mutations in the CYP11B1 gene and is the second major form of congenital adrenal hyperplasia associated with hypertension.

Objective

The objective of this study was to screen the CYP11B1 gene for mutations in one Vietnamese male suffering from congenital adrenal hyperplasia.

Patient

The patient (46,XY) had congenital adrenal hyperplasia. The clinical manifestations presented precocious puberty, hyper-pigmentation and high blood pressure at 4 years.

Results

The patient was a homozygous carrier of a novel mutation located in exon 7 containing a premature stop codon instead of tyrosine at 395 (p.Y395X).

Conclusion

We have identified a novel mutant of the CYP11B1 gene in one Vietnamese family associated with phenotypes of congenital adrenal hyperplasia. The mutant gene p.Y395X produces a truncated form of the polypeptide and abolishes the enzyme activities, leading to a severe phenotype of congenital adrenal hyperplasia.