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A de Kok

Publications and source records attributed to A de Kok.

At least 55 records · Page 3Linked to original sources
PubMed1990

The 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii. 2. Molecular cloning and sequence analysis of the gene encoding the succinyltransferase component.

The nucleotide sequence encoding the succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. Previously the cloning in Escherichia coli of the gene encoding lipoamide dehydrogenase from A. vinelandii was reported [Westphal, A.H. & de Kok, A. (1988) Eur. J. Biochem. 172, 299-305]. The 3.2-kb fragment used for the sequence determination contained the main part of the gene encoding succinyltransferase. The complete E2o gene, as well as the gene encoding the 2-oxoglutarate dehydrogenase component, resided on a 14.7-kb fragment from which the 3.2-kb fragment was subcloned. The protein-coding sequence of the gene consists of 1200 bp (400 codons, including the AUG start codon and the UGA stop codon). It is separated from the gene encoding the 2-oxoglutarate dehydrogenase component by 42 bp. No E. coli-like promoter sequence was found. A putative ribosome-binding site is located 9-15 bp upstream from the start codon. No terminator sequences were found downstream of the stop codon. This makes it likely that the three genes of the oxoglutarate dehydrogenase complex are transcribed as a single mRNA transcript analogous to the pyruvate dehydrogenase complex in E. coli. The intact gene was subcloned from the 14.7-kb fragment and brought to high expression under the influence of the vector-encoded lacZ promoter. The similarity with the E. coli enzyme is high with 63% identity. Like the enzyme from E. coli, it consists of a single lipoyl-binding domain, a putative E1- and E3-binding domain and a catalytic domain. The main difference is found in a 31-residue sequence rich in alanine and proline located between the lipoyl domain and the putative E1- and E3-binding domain. This sequence, usually found in acetyltransferases and there identified as a highly mobile region by 1H-NMR, is replaced by a more polar, charged region in the E. coli enzyme.

Acyltransferases↗
PubMed1989

The gene encoding dihydrolipoyl transacetylase from Azotobacter vinelandii. Expression in Escherichia coli and activation and isolation of the protein.

The gene encoding the dihydrolipoyl transacetylase (E2) component from Azotobacter vinelandii has been cloned in Escherichia coli. High expression of the gene was found when the cells were grown for more than 14 h. The E2 produced was partially active, varying 10 and 90% in different experiments. By limited proteolysis of the protein it was shown that the catalytic domain was incorrectly folded, caused by formation of intermolecular or intramolecular S-S bridges. The enzyme was fully activated after unfolding in 2.5 M guanidine hydrochloride containing 2 mM dithiothreitol, followed by refolding by dialysis. Active E2 was isolated in a simple three-step procedure. It possessed a specific activity in the same order as that found after isolation of E2 from purified pyruvate dehydrogenase complex from A. vinelandii. Active E2 comprises about 7% of the total soluble cellular protein in the E. coli clone. By genetic manipulation, deletion mutants of E2 were created, one encoding the lipoyl domain and the N-terminal half of the pyruvate-dehydrogenase (E1)- and lipoamide-dehydrogenase (E3)-binding domain, the other encoding the catalytic domain and the C-terminal half of the E1- and E3-binding domain. In E. coli expression of both mutants was observed.

Acetyltransferases↗
PubMed1988

Mobile sequences in the pyruvate dehydrogenase complex, the E2 component, the catalytic domain and the 2-oxoglutarate dehydrogenase complex of Azotobacter vinelandii, as detected by 600 MHz 1H-NMR spectroscopy.

600 MHz 1H-NMR spectroscopy demonstrates that the pyruvate dehydrogenase complex of Azotobacter vinelandii contains regions of the polypeptide chain with intramolecular mobility. This mobility is located in the E2 component and can probably be ascribed to alanine-proline-rich regions that link the lipoyl subdomains to each other as well as to the E1 and E3 binding domain. In the catalytic domain of E2, which is thought to form a compact, rigid core, also conformational flexibility is observed. It is conceivable that the N-terminal region of the catalytic domain, which contains many alanine residues, is responsible for the observed mobility. In the low-field region of the 1H-NMR spectrum of E2 specific resonances are found, which can be ascribed to mobile phenylalanine, histidine and/or tyrosine residues which are located in the E1 and E3 binding domain that links the lipoyl domain to the catalytic domain. In the 1H-NMR spectrum of the intact complex, these resonances cannot be observed, indicating a decreased mobility of the E1 and E3 binding domain.

Acetyltransferases↗
PubMed1988

Time-resolved fluorescence studies on the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

The dihydrolipoyl transacetylase (E2) component of A. vinelandii PDC and its lipoyl domain shows similar dynamic properties as revealed with fluorescence anisotropy decay of lipoyl-bound IAANS. The lipoyl domain (32.6 kDa), containing three almost identical subdomains shows a mode of rotation characteristic for a protein of about 30 kDa. A similar rotation is found in E2, indicating an independent rotational mobility of the whole domain in the multimeric E2 core (1.6 MDa). No independent rotation of a single lipoyl subdomain (10 kDa) is observed. The E1 component, in contrast to the E3 component, shows interaction with the lipoyl domain.

Acetyltransferases↗
PubMed1988

The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Molecular cloning and sequence analysis.

The gene encoding the dihydrolipoyltransacetylase component (E2) of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been cloned in Escherichia coli. A plasmid containing a 2.8-kbp insert of A. vinelandii chromosomal DNA was obtained and its nucleotide sequence determined. The gene comprises 1911 base pairs, 637 codons excluding the initiation codon GUG and stop codon UGA. It is preceded by the gene encoding the pyruvate dehydrogenase component (E1) of pyruvate dehydrogenase complex and by an intercistronic region of 11 base pairs containing a good ribosome binding site. The gene is followed downstream by a strong terminating sequence. The relative molecular mass (64913), amino acid composition and N-terminal sequence are in good agreement with information obtained from studies on the purified enzyme. Approximately the first half of the gene codes for the lipoyl domain. Three very homologous sequences are present, which are translated in three almost identical units, alternated with non-homologous regions which are very rich in alanyl and prolyl residues. The N-terminus of the catalytic domain is sited at residue 381. Between the lipoyl domain and the catalytic domain, a region of about 50 residues is found containing many charged amino acid residues. This region is characterized as a hinge region and is involved in the binding of the pyruvate dehydrogenase and lipoamide dehydrogenase components. The homology with the dihydrolipoyltransacetylase from E. coli is high: 50% amino acid residues are identical.

Acetyltransferases↗
PubMed1988

Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene.

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9-23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded form SDS gel electrophoresis of the cell-free extract, stained for protein or used for Western blotting. After subcloning of the 14.7-kb insert of this plasmid the structural gene could be located on a 3.2-kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined. The protein-coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome-binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.

Amino Acid Sequence↗
PubMed1987

The domain structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

Limited proteolysis with trypsin has been used to study the domain structure of the dihydrolipoyltransacetylase (E2) component of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Two stable end products were obtained and identified as the N-terminal lipoyl domain and the C-terminal catalytic domain. By performing proteolysis of E2, which was covalently attached via its lipoyl groups to an activated thiol-Sepharose matrix, a separation was obtained between the catalytic domain and the covalently attached lipoyl domain. The latter was removed from the column after reduction of the S-S bond and purified by ultrafiltration. The lipoyl domain is monomeric with a mass of 32.6 kDa. It is an elongated structure with f/fo = 1.62. Circulair dichroic studies indicates little secondary structure. The catalytic domain is polymeric with S20.w = 17 S and mass = 530 kDa. It is a compact structure with f/fo = 1.24 and shows 40% of the secondary structure of E2. The cubic structure of the native E2 is retained by this fragment as observed by electron microscopy. Ultracentrifugation in 6 M guanidine hydrochloride in the presence of 2 mM dithiothreitol yields a mass of 15.8 kDa. An N-terminal sequence of 36 amino acids is homologous with residues 370-406 of Escherichia coli E2. The catalytic domain possesses the catalytic site, but in contrast to the E. coli subunit binding domain the pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3) binding sites are lost during proteolysis. From comparison with the E. coli E2 sequence a model is presented in which the several functions, such as lipoyl domain, the E3 binding site, the catalytic site, the E2/E2 interaction sites, and the E1 binding site, are indicated.

Acetyltransferases↗
PubMed1987

Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time-resolved flavin fluorescence.

Subnanosecond-resolved fluorescence measurements of the FAD bound in glutathione reductase and lipoamide dehydrogenase revealed characteristic differences in dynamic properties of both enzymes, which are considered to have common structural features. The flavin fluorescence in glutathione reductase is quenched mainly via a dynamic mechanism, in agreement with enhanced flexibility of the flavin as inferred from rapid depolarization of the fluorescence.

Allosteric Site↗
PubMed1987

Gas and liquid chromatography of hydroxybiphenyls, chlorinated hydroxybiphenyls and several types of halogenated derivatives. I. Capillary gas chromatography and mass spectrometry.

For 8 hydroxybiphenyls and 19 chlorinated hydroxybiphenyls derivatization reactions with 6 electrophilic reagents--viz., 4 perfluoro anhydrides, pentafluorobenzoyl chloride and tert.-butylpentafluorophenylmethylchlorosilane--have been studied to evaluate their potential for capillary gas chromatography with electron-capture detection. For some of the classes of derivatives, mass spectrometric data are presented.

Biphenyl Compounds↗
PubMed1985

Hybrid pyruvate dehydrogenase complexes reconstituted from components of the complexes from Escherichia coli and Azotobacter vinelandii.

The pyruvate dehydrogenase complex of Escherichia coli was isolated in a simple three-step procedure. Its chain stoichiometry, determined by trinitrobenzoate modification was found to be 1.4 E1:1 E2:0.6 E3. It was reproducible within 10% from preparation to preparation. The E. coli complex was resolved by chromatography on activated thiol Sepharose. Reconstitution of activity yielded a stoichiometry of 1.0 E1:1 E2:0.5 E3. The optimum binding stoichiometry of E1E2 and E2E3 subcomplexes was determined by sedimentation experiments and found to be 2.0 E1:1 E2 and 2.5 E3:1 E2, respectively. Competition between E1 and E3 was observed in the binding experiments, but not in the kinetic experiments. Hybrid active complexes could be reconstituted from either an E1E2 subcomplex from Azotobacter vinelandii and the E3 component from E. coli or from E2E3 subcomplex from E. coli and the E1 component from A. vinelandii. Low activity and weak binding was observed when E1 from E. coli was recombined with an E2E3 subcomplex from A. vinelandii or when E3 from A. vinelandii was recombined with an E1E2 subcomplex from E. coli. The association behaviour and stoichiometry of the reconstituted complexes is determined by the nature of the E2 component. The formation of hybrid complexes indicates a considerable structural similarity between the complexes from both sources, despite the differences in size and stoichiometry.

Azotobacter↗
PubMed1984

The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Does a unifying model exist for the complexes from gram-negative bacteria?

An improved purification procedure of the pyruvate dehydrogenase complex of Azotobacter vinelandii is described. This procedure minimizes losses of components and results in the isolation of the pure complex with a specific activity of 15-19 U/mg and an overall yield of 40%. The chain ratio of the three components was determined by covalent modification of the lysine residues with trinitrobenzene sulfonic acid, followed by separation of the components on sodium dodecyl sulfate gels. These determinations yielded an average chain ratio of 1.3:1:0.5 for E1:E2:E3 respectively. Based on E2 this corresponds with a minimum molecular mass of approximately 216 kDa. Because the molecular mass of the complex has been determined previously to be 800 +/- 50 kDa, it is concluded that the complex as isolated from A. vinelandii is based on a tetramer of E2 chains. The complex can be resolved into its individual components, which can be recombined to yield a fully active complex. Titration of E2E3 subcomplexes with E1 resulted in maximum complex activity at an E1/E2 ratio of 1.5-1.6. Similar titrations of E1E2 subcomplexes with E3 resulted in maximum activity at an E3/E2 ratio of 0.45-0.55. From these experiments it is concluded that the complex has maximum activity with a composition of three E1 dimers, one E2 tetramer and one E3 dimer. With excess of either E1 or E3 a decrease in activity is observed which indicates competition between these components for binding sites on E2. As shown before [Bosma, H.J., de Kok, A., Markwijk, B.W., and Veeger, C. (1984) Eur. J. Biochem. 140, 273-280], the isolated E2 component is composed of 32 peptide chains of 66 kDa each. Upon addition of E1 or E3, E2 dissociated into tetramers. Dissociation is complete upon the addition of four E1 dimers of four E3 dimers per E2 tetramer. Addition of E1 to saturated E2E3 subcomplex or E3 to saturated E1E2 subcomplex did not result in extra binding but rather in displacement of bound E3 or E1 respectively. It is therefore concluded that the binding sites of E1 and E3 to the E2 chains are either identical or so closely spaced that steric hindrance prevents simultaneous binding of both components. A model is presented based on the cubic structure of the isolated E2 component. In this model the 32 E2 peptide chains are arranged in tetramers in the corners of the cube. This model is discussed in connection with the existing model for the Escherichia coli complex.

Amino Acids↗
PubMed1984

The use of various chromatographic techniques for the determination of phenylurea herbicides and their corresponding anilines in environmental samples. II. Applications.

In earlier work, various strategies have been developed for the trace-level determination of phenylurea herbicides and the anilines which are their main degradation products. They include catalytic hydrolysis of the phenylureas on silica, liquid chromatographic fractionation of complex mixtures of herbicides and anilines, derivatization of anilines and herbicides with electron-capture-sensitive reagents, and final analysis by means of capillary gas chromatography. In the present paper, the application of these principles to trace-level analysis of surface water, soil and crop samples is demonstrated.

Aniline Compounds↗
PubMed1982

An evaluation of chromatographic methods for the analysis of polychlorinated terphenyls in environmental samples.

Gas chromatography (GC) with packed and capillary columns, high-performance liquid chromatography (HPLC) and thin-layer chromatography have been used to characterize various commercially available mixtures of polychlorinated terphenyls (PCTs). Besides, UV absorption and mass spectrometry data have been collected, and the behaviour of PCTs upon perchlorination to the tetradecachloroterphenyls has been studied. Using GC, the PCT content of a number of paper and sewage sludge samples has been determined. The application of perchlorination/GC as method of analysis often yields unreliable, i.e., too high, results compared with the direct GC pattern-comparison method. In the case of paper samples, hydrogenated terphenyls appear to be the principal interfering compounds.

Chromatography, Gas↗
PubMed1977

Analysis of polybrominated biphenyls.

High-performance liquid chromatography, gas chromatography-mass spectrometry, ultraviolet spectrometry and nuclear magnetic resonance spectrometry have been used to characterize the behaviour of a number of individual brominated biphenyls and to assess the composition of several mixtures of these compounds. The dependence of retention and spectral characteristics on the bromine content of the substituted biphenyls is discussed and the behaviour of polybrominated biphenyls is compared with that of polychlorinated biphenyls. The applicability of high-performance liquid chromatography to photodegradation studies is demonstrated.

Biphenyl Compounds↗