Biomedical subjects

C Veeger

Publications and source records attributed to C Veeger.

At least 37 records · Page 2Linked to original sources

PubMed1993

Kinetics of cytochromes P-450 IA1 and IIB1 in reconstituted systems with dilauroyl- and distearoyl-glycerophosphocholine.

In the present study the effect of changing the fatty acyl moiety of phosphatidylcholine from dilauroyl to distearoyl on the kinetic parameters of O-dealkylation of alkoxyresorufins and ethoxycoumarin dependent on reconstituted cytochromes P-450 IA1 and IIB1 has been investigated. The results demonstrate that (a) the maximum rate of O-dealkylation (V) for both P-450 enzymes was about two times higher in the L-alpha-dilauroyl-sn-glycero-3-phosphocholine (Lau2GroPCho) system and (b) changes in the fatty acyl moiety of phosphatidylcholine (acyl2GroPCho) from dilauroyl to distearoyl affected the apparent Km for the substrate (Kms) of P-450 IA1 and IIB1 in a different way. In addition, (c) the kinetic parameters appeared to be dependent on the acyl2GroPCho/P-450 ratio and a change in this ratio affected the kinetic parameters of P-450 IA1 and IIB1 in a different manner. From these last two observations it was concluded that the mechanism by which phospholipids influence P-450-IIB1-dependent O-dealkylation of ethoxycoumarin is different from that by which they influence P-450-IIB1-dependent O-dealkylation of this substrate. Furthermore, the results of the present study demonstrate that the increase in the rate of O-dealkylation of ethoxycoumarin, reported in the literature for reconstituted systems in the presence of Lau2GroPCho, results from an effect of Lau2GroPCho on both the Kms and the V. In a number of additional experiments possible mechanisms underlying the observed differential effect of Lau2GroPCho and Ste2GroPCho on the Kms and V of P-450 IA1 and IIB1 were investigated. This was done by studying the effect of the two acyl2GroPCho species on the kinetic parameters of some of the different steps of the P-450 cycle, namely substrate binding, oxygen binding and the rate of electron transfer. The results demonstrate an influence of Lau2GroPCho and Ste2GroPCho on (a) substrate binding to cytochrome P-450, (b) the affinity of cytochromes P-450 for NADPH-cytochrome reductase and thus on (c) the electron flow through the reconstituted system. Based on the results from these experiments it was concluded that the increased V of P-450 IA1 and IIB1 in the presence of Lau2GroPCho compared to the systems with Ste2GroPCho was at least in part due to an increased affinity of both P-450 enzymes for NADPH-cytochrome reductase in the presence of Lau2GroPCho compared to Ste2GroPCho.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Regioselectivity of cytochrome P-450 catalyzed hydroxylation of fluorobenzenes predicted by calculated frontier orbital substrate characteristics.

In the present study, a hypothesis is presented for the prediction of the regioselectivity of cytochrome P-450 catalyzed hydroxylation of fluorobenzenes. The regioselectivity of the in vivo hydroxylation of fluorobenzene, 1,2-difluorobenzene, 1,3-difluorobenzene, 1,2,3-triluorobenzene, and 1,2,4-triflurobenzene could be predicted within 6% accuracy on the basis of the substrate's frontier orbital characteristics for electrophilic attack. The in vivo regioselectivity of the hydroxylation of fluorobenzene was not significantly influenced by changes in the cytochrome P-450 enzyme pattern. This implies that the regioselectivity is not predominantly determined by the juxtaposition of the relatively small substrates in the active sites of the cytochrome P-450s catalyzing the reaction. Additional in vitro experiments using 1,2-difluorobenzene as the model substrate demonstrated that minor factors influencing the regioselectivity and possibly responsible for the 6% deviation from the calculated values in in vivo experiments might be (i) the influence of biotransformation routes occurring in vivo but not of importance in in vitro microsomal incubations and (ii) a small variation due to influences of the contribution of various cytochrome P-450 enzymes. On the basis of the results obtained, it is concluded that the aromatic hydroxylation of fluorobenzenes proceeds through an initial electrophilic attack of (FeO)3+ on the aromatic substrate, and not through initial electron abstraction followed by attack of the (FeO)2+ species on the substrate radical cation. The fact that the regioselectivity observed could be predicted and/or explained by the site of initial (FeO)3+ attack also argues against epoxides as important intermediates in the formation of phenol metabolites from fluorobenzenes.

Animals↗

PubMed1993

Dissociation of lactate dehydrogenase in aqueous and reversed micellar solutions. A time-resolved polarized fluorescence study.

Dissociation behavior of lactate dehydrogenase from hog muscle, both in aqueous solution and reversed micelles of sodium bis(2-ethylhexyl sulfosuccinate) in octane was studied using time-resolved polarized fluorescence spectroscopy. It was found that, in aqueous solutions, the enzyme underwent partial dissociation with the formation of isolated subunits at enzyme concentrations below 8 nM. Dissociation of the enzyme also took place upon entrapment of lactate dehydrogenase into reversed micelles under conditions of low surfactant hydration, when micelles were not large enough to accommodate a whole protein molecule.

Animals↗

PubMed1993

Flavin dynamics in reduced flavodoxins. A time-resolved polarized fluorescence study.

The time-resolved fluorescence and fluorescence anisotropy characteristics of reduced flavin mononucleotide in solution as well as bound in flavodoxins isolated from the bacteria Desulfovibrio gigas, Desulfovibrio vulgaris, Clostridium beijerinckii MP and Megasphaera elsdenii have been examined. All fluorescence and fluorescence anisotropy decays were analyzed by two different methods: (a) least-squares fitting with a sum of exponentials and (b) the maximum entropy method to yield distributed lifetimes and correlation times. The results of both approaches are in excellent agreement. The fluorescence decay of the free as well as protein-bound reduced flavin chromophore is made up of three components. The shortest component proves to be relatively sensitive to the environment and can therefore be used as a diagnostic tool to probe the microenvironment of the reduced isoalloxazine ring system. The other two longer fluorescence lifetime components are insensitive to the chromophore environment and seem therefore to be related to intrinsic, photophysical properties of the reduced chromophore. Fluorescence anisotropy decays show that the flavin mononucleotide in all four reduced flavodoxins is immobilized within the protein matrix, as indicated by the recovery of a single rotational correlation time, reflecting the rotation of the whole protein. No indications are found that rapid structural fluctuations occur in reduced flavodoxins, and the mechanism of electron transfer from flavodoxin to other redox proteins seems to involve immobilized reduced flavin.

Clostridium↗

PubMed1993

Comparison of the dynamic structure of alpha-chymotrypsin in aqueous solution and in reversed micelles by fluorescent active-site probing.

A highly fluorescent anthraniloyl (Ant) group was covalently attached to the active site of alpha-chymotrypsin (CT), probably at Ser195. Ant-CT is stable at neutral pH for months, allowing a detailed fluorescence study of Ant-CT as a model protein to investigate its physical properties in 0.1 M Tris/HCl, pH 8.2, and in reversed micelles of n-octane, 0.1 M Tris/HCl, pH 8.2, and sodium bis(2-ethylhexyl)sulfosuccinate (AOT). Steady-state fluorescence measurements of the progressive red-shift of the center of gravity of the emission band as function of degree of hydration, wo, defined as [H2O]/[AOT], indicate that the average polarity in the vicinity of the probe is approaching that of bulk water at wo > 12. Time-resolved fluorescence measurements of Ant-CT in water and in reversed micelles showed that the active site has different properties in reversed micelles compared to those in water. Some specific changes at very low water content (0.6 < wo < 5) can be observed, which correlate with enzyme activity measurements in the same wo region (unpublished results). These effects are, for instance, significant changes in the average fluorescence lifetime and the internal flexibility of the probe. The overall rotational-correlation time of the enzyme in AOT reversed micelles seems to be independent on wo (5 < wo < 29), which suggests that the enzyme creates its own micelle.

Animals↗

PubMed1993

Enzyme inactivation and protection during entrapment in reversed micelles.

Lactate dehydrogenase (LDH) was found to lose rapidly its catalytic activity during the process of entrapment in hydrated reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in octane. It was demonstrated that this inactivation was caused by the surfactant which penetrated into the injected enzyme-containing aqueous bulk phase during the short time of mechanical stirring needed to convert the initial biphasic mixture into a monophasic reversed micellar solution. The unfavorable inactivation phenomenon could be efficiently eliminated by the addition of either NADH or pyruvate into the enzyme stock solution prior to its injection into AOT solution in octane. The catalytic activity of substrate-protected LDH in AOT reversed micelles increased with increasing water content of the micellar system, reaching the maximal level above wo = 30 when aqueous inner cores of reversed micelles grew large enough to allow unrestricted accommodation of the enzyme molecule. It is suggested that the employment of substrate-protected enzymes could represent a generally useful approach for producing highly efficient enzyme-containing reversed micellar reaction systems.

Catalysis↗

PubMed1992

Overproduction of prismane protein in Desulfovibrio vulgaris (Hildenborough): evidence for a second S = 1/2-spin system in the one-electron reduced state.

The gene encoding the prismane protein from Desulfovibrio vulgaris (Hildenborough) was inserted into broad-host-range vector pSUP104. The recombinant plasmid, pJSP104, was transferred to D. vulgaris by conjugal plasmid transfer. In the transconjugant D. vulgaris cells the prismane protein was 25-fold overproduced. The overproduced prismane protein was characterized by molecular mass, isoelectric point, iron content and spectroscopical properties. Both the iron content and the ultraviolet/visible spectrum are identical to the wild-type protein indicating that iron incorporation in the overproduced protein is complete. EPR spectra of the dithionite-reduced form of the overproduced protein indicated that the Fe-S cluster might occur in a similar structure as found in inorganic model compounds containing a [6Fe-6S] prismane core. The as-isolated overproduced protein showed the presence of a second S = 1/2 spin system that was also detected in the corresponding prismane protein from D. desulfuricans (ATCC 27774), but not in the protein from wild-type D. vulgaris. This additional signal was irreversibly transformed to the 'wild-type' high-spin and low-spin systems upon two reduction/re-oxidation cycles. It is shown that the EPR spectroscopy of the overproduced prismane protein is very similar to that of the D. desulfuricans enzyme and, with the exception of the second S = 1/2 spin system, to that of the prismane protein from wild-type D. vulgaris. Contrary to claims for the D. desulfuricans protein, it is shown here that all data can be fully explained assuming a single [6Fe-6S] cluster, that might be titrated into four different redox states and occurs in up to three different spin systems in the one-electron reduced state.

Bacterial Proteins↗

PubMed1992

Redox properties of the iron-sulfur clusters in activated Fe-hydrogenase from Desulfovibrio vulgaris (Hildenborough).

The periplasmic Fe-hydrogenase from Desulfovibrio vulgaris (Hildenborough) contains three iron-sulfur prosthetic groups: two putative electron transferring [4Fe-4S] ferredoxin-like cubanes (two F-clusters), and one putative Fe/S supercluster redox catalyst (one H-cluster). Combined elemental analysis by proton-induced X-ray emission, inductively coupled plasma mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy and colorimetry establishes that elements with Z > 21 (except for 12-15 Fe) are present in 0.001-0.1 mol/mol quantities, not correlating with activity. Isoelectric focussing reveals the existence of multiple charge conformers with pI in the range 5.7-6.4. Repeated re-chromatography results in small amounts of enzyme of very high H2-production activity determined under standardized conditions (approximately 7000 U/mg). The enzyme exists in two different catalytic forms: as isolated the protein is 'resting' and O2-insensitive; upon reduction the protein becomes active and O2-sensitive. EPR-monitored redox titrations have been carried out of both the resting and the activated enzyme. In the course of a reductive titration, the resting protein becomes activated and begins to produce molecular hydrogen at the expense of reduced titrant. Therefore, equilibrium potentials are undefined, and previously reported apparent Em and n values [Patil, D. S., Moura, J. J. G., He, S. H., Teixeira, M, Prickril, B. C., DerVartanian, D. V., Peck, H. D. Jr, LeGall, J. & Huynh, B.-H. (1988) J. Biol. Chem. 263, 18,732-18,738] are not thermodynamic quantities. In the activated enzyme an S = 1/2 signal (g = 2.11, 2.05, 2.00; 0.4 spin/protein molecule), attributed to the oxidized H cluster, exhibits a single reduction potential, Em,7 = -307 mV, just above the onset potential of H2 production. The midpoint potential of the two F clusters (2.0 spins/protein molecule) has been determined either by titrating active enzyme with the H2/H+ couple (E,m = -330 mV) or by dithionite-titrating a recombinant protein that lacks the H-cluster active site (Em,7.5 = -340 mV). There is no significant redox interaction between the two F clusters (n approximately 1).

Copper↗

PubMed1992

Differential cumene hydroperoxide sensitivity of cytochrome P-450 enzymes IA1 and IIB1 determined by their way of membrane incorporation.

The cytochrome P-450-dependent O-dealkylation of alkoxyresorufins was used to study the effect of cumene hydroperoxide on cytochrome P-450 IIB1 and IA1 in microsomal and reconstituted systems. In liver microsomal systems from respectively phenobarbital and 3-methylcholanthrene pretreated male Wistar rats, cytochrome P-450 IIB1-dependent pentoxyresorufin-O-dealkylation appeared to be more sensitive to cumene hydroperoxide treatment than cytochrome P-450 IA1-dependent ethoxyresorufin-O-dealkylation. This phenomenon was also observed when the cumene hydroperoxide sensitivity of P-450 IIB1 and IA1 was studied in an isosafrole pretreated rat liver microsomal system. The decrease in alkoxy-O-dealkylating activities appeared to proceed by destruction of the cytochrome P-450 component of the enzyme system. Purification and reconstitution of the enzyme system components in a system in which the isolated proteins were not incorporated into a membrane resulted in the disappearance of the difference in sensitivity between the two P-450 enzymes. However, in a reconstituted system with membrane incorporated proteins, again cytochrome P-450 IIB1 expressed a higher sensitivity towards cumene hydroperoxide than cytochrome P-450 IA1. From this it was concluded that the differential cumene hydroperoxide sensitivity of cytochrome P-450 IIB1 and IA1 is not caused by an intrinsic difference in their sensitivity but by a differential effect of membrane incorporation on their cumene hydroperoxide sensitivity.

Animals↗

PubMed1992

The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio vulgaris (Hildenborough).

The gene encoding a protein containing a putative [6Fe-6S] prismane cluster has been cloned from Desulfovibrio vulgaris (Hildenborough) and sequenced. The gene encodes a polypeptide composed of 553 amino acids (60,161 Da). The DNA-derived amino acid sequence was partly confirmed by N-terminal sequencing of the purified protein and of fragments of the protein generated by CNBr cleavage. Furthermore, the C-terminal sequence was verified by digestion with carboxypeptidases A and B. The polypeptide contains nine Cys residues. Four of these residues are gathered in a Cys-Xaa2-Cys-Xaa7-Cys-Xaa5-Cys motif located towards the N-terminus of the protein. No relevant sequence similarity was found with other proteins, including those with high-spin Fe-S clusters (nitrogenase, hydrogenase), with one significant exception: the stretch containing the first four Cys residues spans two submotifs, Cys-Xaa2-Cys and Lys-Gly-Xaa-Cys-Gly, separated by 11 residues, that are also present in high-spin Fe-S cluster containing CO dehydrogenase. Western-blot analysis demonstrates cross-reactivity of antibodies raised against the purified protein both in Desulfovibrio strains and other sulfate-reducing bacteria. Hybridization of the cloned gene with genomic DNA of several other Desulfovibrio species indicates that homologous sequences are generally present in the genus Desulfovibrio.

Amino Acid Sequence↗

PubMed1992

The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio desulfuricans (ATCC 27774).

The gene encoding a protein containing a novel iron sulfur cluster ([6Fe-6S]) has been cloned from Desulfovibrio desulfuricans ATCC 27774 and sequenced. An open reading frame was found encoding a 545 amino acid protein (M(r) 58,496). The amino acid sequence is highly homologous with that of the corresponding protein from D. vulgaris (Hildenborough) and contains a Cys-motif that may be involved in coordination of the Fe-S cluster.

Amino Acid Sequence↗

PubMed1992

Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Kinetics of wild-type and mutated enzymes.

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced with other residues. His450, the active-site base, was replaced with Ser, Tyr or Phe. Pro451, from X-ray analysis found to be in cis conformation positioning the backbone carbonyl of His450 close to N3 of the flavin, was changed to Ala. Glu455, from X-ray analysis expected to be involved in modulating the pKa of the base (His450), was replaced with Asp and Gln. The general conclusion is that mutation of the His-Glu diad impairs intramolecular electron transfer between the disulfide/dithiol and the FADH-/FAD. The wild-type enzyme functions according to a ping-pong mechanism in the physiological reaction in which the formation of NADH is rate-limiting. Above pH 8.0 the enzyme is strongly inhibited by the product NADH. The pH dependence of the steady-state kinetics using the NAD+ analog 3-acetylpyridine adenine dinucleotide (AcPyAde+) reveals a pKa of 8.1 in the pKm AcPyAde+ plot indicating that this pKa is related to the deprotonation of His450 [Benen, J., Berkel van, W., Zak, Z., Visser, T., Veeger, C. & Kok de, A. (1991) Eur. J. Biochem. 202, 863-872] and to the inhibition by NADH. The mutations considerably affect turnover. Enzymes with the mutations Pro451----Ala, His450----Phe and His450----Tyr appear to be almost inactive in both directions. Enzyme His450----Ser is minimally active, V at the pH optimum being 0.5% of wild-type activity in the physiological reaction. Rapid reaction kinetics show that for the His450-mutated enzymes the reductive half reaction using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared to the wild-type enzyme. For enzyme Pro451----Ala it is concluded that the loss of activity is due to over-reduction by Lip(SH)2 and NADH. The Glu455-mutated enzymes are catalytically competent but show strong inhibition by the product NADH (enzyme Glu455----Asp more than Glu455----Gln). The inhibition can largely be overcome by using AcPyAde+ instead of NAD+ in the physiological reaction. The rapid reaction kinetics obtained for enzymes Glu455----Asp and Glu455----Gln deviate from the wild-type enzyme. It is concluded that this difference is due to cooperativity between the active sites in this dimeric enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter vinelandii↗

PubMed1992

Lipoamide dehydrogenase from Azotobacter vinelandii. The role of the C-terminus in catalysis and dimer stabilization.

The 10 C-terminal residues are not visible in the crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii, but can be observed in the crystal structures of the lipoamide dehydrogenases from Pseudomonas putida and Pseudomonas fluorescens. In these structures, the C-terminus folds back towards the active site and is involved in interactions with the other subunit. The function of the C-terminus of lipoamide dehydrogenase from A. vinelandii was studied by deletion of 5, 9 and 14 residues, respectively. Deletion of the last 5 residues does not influence the catalytic properties and conformational stability (thermoinactivation and unfolding by guanidinium hydrochloride). Removal of 9 residues results in an enzyme (enzyme delta 9) showing decreased conformational stability and high sensitivity toward inhibition by NADH. These features are even more pronounced after deletion of 14 residues (enzyme delta 14). In addition Tyr16, conserved in all lipoamide dehydrogenases sequenced thus far, and shown from the other structures to be likely to be involved in subunit interaction, was replaced by Phe and Ser. Mutation of Tyr16 also results in a strongly increased sensitivity toward inhibition by NADH. The conformational stability of both Tyr16-mutated enzymes is comparable to enzyme delta 9. The results strongly indicate that a hydrogen bridge between tyrosine of one subunit (Tyr16 in the A. vinelandii sequence) and histidine of the other subunit (His470 in the A. vinelandii sequence), exists in the A. vinelandii enzyme. In the delta 9 and delta 14 enzymes this interaction is abolished. It is concluded that this interaction mediates the redox properties of the FAD via the conformation of the C-terminus containing residues 450-470.

Azotobacter vinelandii↗

PubMed1992

The pH in reversed micelles as imposed by the dihydrogen/proton redox couple and indicated by viologens and cytochrome c3 using hydrogenase as redox catalyst.

The pH values in reversed micelles were measured, making use of the hydrogenase enzyme as redox catalyst short-circuiting the viologen oxidized/semiquinone redox states. The hydrogenases from Desulfovibrio vulgaris (Hildenborough) and from Megasphaera elsdenii were applied. The observed pH values in reversed micelles were not dependent on the type of hydrogenase. Two cationic [cetyltrimethylammonium bromide and dodecylammonium propionate (DAP)] and two anionic sodiumdodecyl sulphate, sodium di(ethylhexyl)sulfosuccinate types of reversed micelles were used in combination with viologens having distinguishable valencies. It was observed that, in the cationic-reversed micelles, the dissociation constant for the semiquinone dimer had about the same value as compared to bulk water, while this value was significantly higher in the anionic-reversed micelles. Furthermore, the dissociation constant was independent of the concentration of viologen semiquinone in the reversed micelle, indicating that exchange kinetics are faster than the dimerisation process. With the exception of DAP, a linear relation exists, pH = a.pHrm + b, between the pH of the bulk water and the pH as measured in the reversed micelle (pHrm). In all these cases the value of a is smaller than unity, the value of b ranges between 1.6-2.7. For DAP the pHrm is always around 7. In DAP-reversed micelles, the counter-ion propionate probably serves as an internal buffer. Using cytochrome c3 as pH indicator in combination with N,N'-di(3-aminopropyl)-4,4'-bipyridinium)4+ to take care of electron transfer, in cetyltrimethylammonium-bromide-reversed micelles the pHrm is about the same as indicated by the viologen; in SDS-reversed micelles the pHrm is always lower than that indicated by N,N'-di(3-aminopropyl)4,4'-pyridinium4+. In contrast to cytochrome c3 from D. vulgaris, which in reversed micelles cannot become reduced directly by its D. vulgaris hydrogenase, the hydrogenase of M. elsdenii is able to reduce its ferredoxin directly.

Bromides↗

PubMed1992

Purification and biochemical characterization of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough).

A novel iron-sulfur protein has been isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. Analysis of the metal and acid-labile sulfur content of the protein revealed the presence of 6.3 +/- 0.4 Fe/polypeptide and 6.2 +/- 0.7 S2-/polypeptide. Non-iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S = 1/2 [6Fe-6S]3+ prismane-like EPR signal in the dithionite-reduced protein, we believe that we have encountered the first example of a prismane-cluster-containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI = 4.9). The ultraviolet/visible spectrum is relatively featureless (epsilon 280 = 81 mM-1.cm-1, epsilon 400 = 25 mM-1.cm-1, epsilon 400,red = 14 mM-1.cm-1). The shape of the protein is approximately globular (S20.w = 4.18 S). The N-terminal amino acid sequence is MFS/CFQS/C QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross-reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.

Amino Acid Sequence↗

PubMed1992

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli.

Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichia coli. In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively. Chimeric E2p components were constructed by exchanging the three domains between E2p from A. vinelandii and E. coli on gene level. The six chimeric E2p proteins were expressed and purified from E. coli TG2. All chimeras were catalytically active, 24-subunit E2p proteins. Interactions of the peripheral components E1p and E3 with the wild-type enzymes from A. vinelandii and E. coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex. A. vinelandii E3 interacts only with those chimeras that contain the A. vinelandii binding domain, whereas E. coli E3 interacts with all chimeras. Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3. Recognition of E1p depends on the origin of both the binding domain and the catalytic domain. E. coli E1p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E. coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E. coli E2p. No binding of E. coli E1p was observed when both domains were of A. vinelandii origin. A. vinelandii E1p recognizes E2p from A. vinelandii and E. coli, but strong interaction required that the binding and catalytic domain were of the same origin. Exchange of lipoyl domains had no effect on the binding properties of the E1p component. These observations confirm previous conclusions, based on site-directed mutagenesis of A. vinelandii E2p [Schulze, E., Westphal, A. H., Boumans, H., and de Kok, A. (1991) Eur. J. Biochem. 202, 841-848], that the binding site for E1p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E. coli E2p. Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A. vinelandii E2p. Dissociation depends on the binding of peripheral components to the E1p-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter vinelandii↗

PubMed1992

Frontier orbital study on the 4-hydroxybenzoate-3-hydroxylase-dependent activity with benzoate derivatives.

Based on molecular orbital computer calculations the present paper provides a new hypothesis for catalytic characteristics of 4-hydroxybenzoate-3-hydroxylase (EC 1.14.13.2). A clear correlation between in kcat for the conversion of a series of 4-hydroxylated substrates and their E(HOMO) leads to the hypothesis that Frontier orbital HOMO characteristics [E(HOMO) and HOMO density on C3] of the substrates are the predominant factor in regulating the fate of a benzoate derivative at the active site of the enzyme. The HOMO characteristics can be used to explain whether a compound will be converted by the enzyme or merely acts as an effector. Furthermore, the hypothesis provides quantitative theoretical support for a catalytic mechanism in which the substrate reacts in its dianionic form and for a mechanism in which the electrophilic attack of the C(4a)-peroxyflavin, or of the hydroxyl radical derived from it, on the benzoate dianion is the rate limiting step in catalysis at pH 8, 25 degrees C. Finally, it is demonstrated that the hypothesis can be used as a basis for the formulation of working hypotheses in future research, investigating the conversion and regioselective orientation of the various possible substrates in the active site of the wild-type 4-hydroxybenzoate-3-hydroxylase, its mutants as well as of various other flavin-dependent aromatic hydroxylases, such as for example 3-hydroxybenzoate-4-hydroxylase (EC 1.14.13.23), 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) and phenol hydroxylase (EC 1.14.13.7).

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1992

Study on the regioselectivity and mechanism of the aromatic hydroxylation of monofluoroanilines.

The in vitro and in vivo metabolism of monofluoroanilines was investigated. Special attention was focused on the regioselectivity of the aromatic hydroxylation by cytochromes P-450 and the mechanism by which this reaction might proceed. The results clearly demonstrate that the in vitro and in vivo regioselectivity of the aromatic hydroxylation by cytochromes P-450 is dependent on the fluoro-substituent pattern of the aromatic aniline-ring. Results from experiments with liver microsomes from differently pretreated rats demonstrate that the observed regioselectivity for the aromatic hydroxylation is not predominantly determined by the active site of the cytochromes P-450. To investigate the underlying reason for the observed regioselectivity, semi-empirical molecular orbital calculations were performed. Outcomes of these calculations show that neither the frontier orbital densities of the LUMO/LUMO + 1 (lowest unoccupied molecular orbital) of the monofluoroanilines nor the spin-densities in their NH. radicals can explain the observed regioselectivities. The frontier orbital densities of the HOMO/HOMO - 1 (highest occupied molecular orbital) of the monofluoroanilines however, qualitatively correlate with the regioselectivity of the aromatic hydroxylation. Based on these results it is concluded that the cytochrome P-450 dependent aromatic hydroxylation of monofluoroanilines does not proceed by hydrogen or electron abstraction from the aniline substrate to give an aniline-NH. radical. The results rather suggest that cytochrome P-450 catalyzed aromatic hydroxylation of monofluoroanilines proceeds by an electrophilic attack of the (FeO)3+ species of cytochrome P-450 on a specific carbon atom of the aromatic aniline-ring.

Aniline Compounds↗