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PubMed1980

Pyridine nucleotide transhydrogenase from Azotobacter vinelandii. Improved purification, physical properties and subunit arrangement in purified polymers.

1. Pyridine nucleotide transhydrogenase from Azotobacter vinelandii was purified with a scaled-up procedure. In a typical purification 500 ml cell-free extract from 200 g cells is loaded on an Ado-2',5'-P2--Sepharose 4B affinity column (20 ml bed volume). After washing, the enzyme is desorbed with 2'AMP at neutral pH and further purified by Sephadex G-200 gel chromatography. The enzyme (10--12 mg) is obtained in 40--60% yield and is homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The homogeneity of the purified enzyme is also apparent from electron microscopy studies, where the enzyme appears as a polydisperse set of polymers without contaminating structures and from fluorescence lifetime studies by the method of single-photon counting. The flavin fluorescence appears to decay with a single lifetime tau = 2.5 ns. The polymeric nature of transhydrogenase can be aptly demonstrated by density gradient centrifugation in the presence of KBr. After centrifuging for 50 h at 160 000 X g and 10 degrees C the enzyme is concentrated in a narrow fluorescent band with buoyant density rho b = 1.305 g cm-3. 3. The arrangement of subunits in the transhydrogenase polymer has been derived from optical diffraction studies of electron micrographs. The polymers are built up from a linear assembly of tetramers. Four subunits are placed in a rhomb with sides of 13.5 mm and an angle of 45 degrees (135 degrees) between the sides. A second tetramer is located staggered on top of the first one. Since a variety of other studies have indicated that the polymers dissociate into octamers under alkaline conditions [Voordouw, G. et al. (1979 Eur. J. Biochem. 98,447--454] we conclude that this smallest functional unit is build up from two tetramers.

Amino Acids↗

PubMed1981

Modification of the thiol residues of pyridine nucleotide transhydrogenase from Azotobacter vinelandii. Activity modulation by the divalent thiol reagent p-aminophenylarsenoxide.

1. Purified pyridine nucleotide transhydrogenase from Azotobacter vinelandii contains three thiol residues as judged by titration with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions. 2. In the native conformation of the transhydrogenase only a single thiol residue is titrated. Modification of this exposed thiol does not influence transhydrogenase activity. 3. The two less exposed thiol residues can be reacted in part with either p-chloromercuribenzoate or N-ethyl-maleimide. Modification of one residue leads to loss of 40-60% of the enzyme activity in both the forward (NAD+ + NADPH leads to NADH + NADP+) and reverse reaction. The strong inhibitory action of phosphate ions on the reverse reaction [Voordouw et al. (1980) Eur. J. Biochem. 107, 337-344] is abolished after treatment with p-chloromercuribenzoate. Reaction with phenylmercurichloride or p-aminophenylmercuriacetate causes a similar activity loss without affecting the inhibitory action of phosphate. 4. The interaction of the divalent thiol inhibitor p-aminophenylarsenoxide with transhydrogenase was found to be reversible and is characterized by an association constant of 6.3 x 10(5) M-1 at 25 degrees C in 50 mM sodium phosphate pH 7.50. This reversibility indicates formation of a cyclic dithiolarsinite derivative with considerable ring strain. The activity of p-aminophenylarsenoxide-transhydrogenase is modulated by phosphate and magnesium ions. The activity of the transhydrogenase . p-aminophenylarsenoxide complex in the forward reaction is inhibited by phosphate and stimulated by magnesium ions. The reverse reaction is not catalyzed by the enzyme-inhibitor complex. 5. The presence of an activity modulating site in transhydrogenase which binds phosphate ions and has the two less exposed thiol residues in close proximity is indicated by the results.

Arsenicals↗

PubMed1983

Binding of MgATP to the nitrogenase proteins from Azotobacter vinelandii.

Binding of MgATP to the MoFe and Fe proteins from Azotobacter vinelandii has been studied. By means of the flow dialysis technique it was demonstrated that one molecule of reduced Fe protein binds one molecule of MgATP, with a dissociation constant of 0.56 +/- 0.11 mM. The oxidized Fe protein binds two molecules of MgATP, with identical intrinsic dissociation constants of 0.29 +/- 0.05 mM. The binding of MgATP to the Fe protein was also studied by equilibrium dialysis. It was found that during dialysis of reduced Fe protein in the presence of MgATP, dithionite was oxidized. Moreover, in the presence of MgATP both reduced and oxidized Fe protein were inactivated during the dialysis. These observations demonstrate that binding of MgATP to the Fe protein can only be measured by a relatively fast method. With the same methods as used for the Fe protein, no binding of MgATP to the MoFe protein of A. vinelandii could be demonstrated. The redox properties of the Fe protein in the presence and absence of MgATP are discussed with respect to the observed binding properties of MgATP for the Fe protein. The implications of these results are discussed with respect to the present models for the interactions between the Fe and MoFe proteins of nitrogenase.

Adenosine Triphosphate↗

PubMed1983

Fully active Fe-protein of the nitrogenase from Azotobacter vinelandii contains at least eight iron atoms and eight sulphide atoms per molecule.

The Fe-protein of the Azotobacter vinelandii nitrogenase enzyme complex contains a variable iron and sulphide content. The iron and sulphide content of the protein is dependent upon the specific activity. Up to a specific activity of 1000 nmol C2H4 produced X min-1 X mg Av-1(2), three iron and three sulphide atoms per molecule Av2 are found. At specific activities above 1000 nmol C2H4 produced X min-1 X mg Av-1(2), a linear relationship between specific activity and iron and sulphide content of Av2 is found. The maximum values found are 8.8 iron atoms and 8.6 sulphide atoms/molecule at a specific activity of 2250 nmol C2H4 produced X min-1 X mg Av-1(2). Also the experimental molar absorption coefficients at 430 nm of the oxidized and reduced forms depend on the specific activity. The highest values found are 15.9 mM-1 cm-1 and 9.1 mM-1 cm-1, respectively. Since occasionally the preparations with specific activities around 3000 nmol X min-1 X mg-1 are isolated which contain more than 10 iron atoms and 11 sulphide atoms per molecule, it cannot be excluded that under certain physiological conditions Av2 contains even more than two [4 Fe-4 S] clusters. The addition of MgATP induces a conformational change in the Fe-protein which results in a higher reactivity with iron chelators. But irrespective of the specific activity, the amount of iron extracted from the protein after addition of MgATP never exceeds four atoms/molecule. The results are discussed with respect to the present molecular model of the Fe-protein.

Azotobacter↗

PubMed1983

On the formation of an oxygen-tolerant three-component nitrogenase complex from Azotobacter vinelandii.

Conditions are defined in which the oxygen-labile nitrogenase components from Azotobacter vinelandii can be protected against oxygen inactivation by the so-called Fe/S protein II. It is demonstrated that oxygen protection can be achieved by complex formation of the three proteins. Complex formation was studied by gel chromatography. Only when the three proteins are in the oxidized state and MgCl2 is present, can an oxygen-tolerant complex be isolated. Quantitative SDS/polyacrylamide gel electrophoresis of such complexes, yielded an average ratio of nitrogenase component 2/nitrogenase component 1 (Av2/Av1) of 2.4 +/- 0.5. Protection by Fe/S protein II was correlated with the amount of [2 Fe-2S] clusters present in the protein and not by the amount of protein. Measurements of the amount of iron and sulfide of Fe/S protein II showed that the iron and sulfide content of the protein was variable. The maximum values found indicate that Fe/S protein II contains two [2Fe-2S] clusters per dimer of 26 kDa. Full protection by Fe/S protein II was obtained with a ratio of Fe/S protein II/Av1 of 1.1 +/- 0.2; the Fe/S protein II containing two [2Fe-2S] clusters per dimer of 26 kDa. When Fe/S protein II contains less [2Fe-2S] clusters, more protein is necessary to obtain full protection. The three-component nitrogenase complex is also oxygen stable in the presence of MgATP or MgADP. Analysis in the ultracentrifuge showed that the major fraction of the reconstituted complex has a sedimentation coefficient centered around 34S. A small fraction (less than 30%) sediments with values centered around 111 S. This suggests an average mass for the oxygen-stable nitrogenase complex of 1.5 MDa. Taking into account the determined stoichiometry of the individual proteins, the molecular composition of the oxygen-stable nitrogenase complex is presumably 4 molecules of AV1,8--12 molecules of aAV2 and 4--6 molecules of Fe/S protein II containing two [2Fe-2S] clusters per dimer of 26 kDa.

Azotobacter↗

PubMed1983

Structure of the Mo-Fe protein component of Azotobacter vinelandii nitrogenase. Analytical ultracentrifugation and electron microscopy studies.

The Mo-Fe protein of nitrogenase from both Azotobacter vinelandii and Klebsiella pneumoniae (Av1 and Kp1, respectively) consists of four subunits of similar, but not identical, relative molecular mass. The hydrodynamic properties of Av1 (sedimentation and diffusion coefficient) and its total relative molecular mass are very similar to those of Kp1 and catalase from bovine liver, a tetramer of four identical subunits. By electron microscopy the Av1, Kp1 and catalase tetramers are seen as protein particles of diameter 9.0-10.0 nm; no details of the subunit structure can be observed. Av1 (but not Kp1) forms regular polymers of variable length at low ionic strength in the presence of MgCl2. The structure of these polymers, of diameter 21.2 nm, is complex. Optical diffraction studies give a smallest repeating distance of 8.4 nm (corresponding to the diameter of the Av1 tetramer) and indicate a four-start helix. The latter structure is incompatible with a flat, square subunit arrangement of the Av1 tetramer as proposed by Stasny et al. [(1974) J. Cell. Biol. 60, 311-316]. We propose, therefore, that the subunit arrangement of the Av1 tetramer is of the tetrahedral type. This has also been proposed for the catalase tetramer from optical diffraction studies of electron micrographs of catalase tubes indicating a 222 symmetry [Kiselev, D. A., De Rosier, N. J. and Klug, A. (1968) J. Mol. Biol. 35, 561-566]. Our proposal is in agreement with the recent finding that Av1 protein crystals belong to the P2(1) space group [Weiniger, M. S. and Mortenson, L. E. (1982) Proc. Natl Acad Sci. USA, 79, 378-380].

Animals↗

PubMed1984

The size of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Association phenomena.

Sedimentation analysis and light-scattering studies indicate that the aggregation state of the pyruvate dehydrogenase complex of Azotobacter vinelandii in 50 mM potassium phosphate (pH 7.0) can be described in terms of a monomer-dimer equilibrium with a dissociation constant of 6.8 microM. The apparent molecular mass of the monomeric particle is 750 000-850 000 Da. The equilibrium is shifted to the monomeric species when pressure is applied on the system. Pressure-jump experiments yielded a relaxation time of about 70 ms. In the presence of 3% poly(ethylene glycol) 6000 and 10 mM MgCl2, further association takes place to a system that can be described in terms of dimer-tetramer-octamer equilibria. Upon applying a pressure of 80 MPa to this system these equilibria are shifted to the dimeric state but some monomer formation cannot be excluded. Release of pressure shows that the relaxation time of the dimer-tetramer equilibrium is less than 5 s, that of tetramer-octamer equilibrium is of the order of minutes. The isolated E2 component has a molecular mass of 2 000 000 +/- 100 000 Da; and thus consists of about 30 E2 peptide chains. Electron micrographs are similar to those of the E2 component of the Escherichia coli complex, which were interpreted as cubic structures with an octagonal symmetry. Upon addition of E1 to the pure E2 component, changes in the assembly occur and mixtures of large (E. coli-like, 22-45 S) and small (A. vinelandii-like, 11-18 S) subcomplexes are obtained. The two forms of the subcomplexes are in slow equilibrium (relaxation time 10-30 min). It is proposed that the E2 tetramer of the intact pyruvate dehydrogenase complex of A. vinelandii is represented by the corner structures of the isolated E2 component.

Azotobacter↗

PubMed1984

Respiratory mutants of Azotobacter vinelandii with elevated levels of cytochrome d.

A method is described for the isolation of respiratory mutants of Azotobacter vinelandii with increased amounts of d-type cytochrome by selecting for the inability to reduce tetrazolium red. Five stable mutants were obtained that had six-fold higher levels of cytochrome d, increased amounts of b-type and lower amounts of o-type and c-type cytochromes than the wild-type strain. Spectral alterations in cytochrome alpha 1 were also observed in the mutants. NADH and succinate oxidase activities of membrane particles were about two fold higher in the mutants compared to the wild-type strain. Ascorbate-N,N,N',N'-tetramethyl-p-phenylene diamine oxidase activity was barely detectable in membrane particles of the mutants. These results are consistent with an increase in the amount of the cytochrome d oxidase branch and a decrease in the amount and activity of the cytochrome o, alpha 1 oxidase branch in the mutants. Growth rates under oxygen-excess conditions and respiratory-linked proton translocation ratios of the mutant and wild-type stains were similar as were the photochemical spectral and kinetic properties of cytochrome d.

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↗

PubMed1985

The importance of quantitative Mössbauer spectroscopy of MoFe-protein from Azotobacter vinelandii.

The Mössbauer spectra of MoFe-protein of Azotobacter vinelandii, as isolated under dithionite and taken at temperatures from 125 K to 175 K, are the sums of four resolved quadrupole doublets. Our results indicate that the currently accepted interpretation of these doublets can be questioned. Our data reduction method converts the Mössbauer transmission spectra to source lineshape deconvolved absorption spectra linear in iron. We used these absorption spectra to determine the stoichiometry of the Fe clusters in MoFe-protein and we obtained much better fits if we assumed that there are four iron atoms in the 'Fe2+, doublet, two iron atoms in the 'S' doublet, twelve iron atoms in the 'D' doublet and sixteen iron atoms in the 'M' doublet. Therefore we propose that the MoFe-cofactor contains one molybdenum and eight iron atoms ('M'). We also argue that none of the previous Mössbauer spectroscopic studies have been performed on the highest-activity preparation now obtainable, nor has there been any study to prove that the Mössbauer spectra are independent of activity. We consider that the Mössbauer spectroscopic studies of the MoFe-protein of nitrogenase are a re-opened and unsolved problem.

Azotobacter↗

PubMed1985

The catalytic activity of nitrogenase in intact Azotobacter vinelandii cells.

The influence of the growth conditions on the concentration of nitrogenase and on the nitrogenase activity, was studied in intact Azotobacter vinelandii cells. It was observed that whole cell nitrogenase activity could be enhanced in two ways. An increase of the growth rate of cells was accompanied by an increase in whole cell nitrogenase activity and by an increase in the concentration of nitrogenase in the cells. The molar ratio of Fe protein:MoFe protein was 1.47 +/- 0.17 and independent of the growth rate. Activity measurements in cell extracts showed that the catalytic activity of the nitrogenase proteins was independent of the growth rate of cells. The second way to increase whole cell nitrogenase activity was to expose cells to excess oxygen. Whole cells were exposed for 2.5 h to an enhanced oxygen-input rate. After this incubation nitrogenase activity was increased without an increase in protein concentration. It is calculated that the catalytic activity of the Fe protein in these cells was 6200 nmol C2H4 formed X min-1 X (mg Fe protein)-1. With these cells and with cells grown at a high growth rate, 50% of the whole cell activity is lost by preparing a cell-free extract. It will be demonstrated that this inactivation is partly caused by the activity measurements in vitro. When dithionite was replaced by flavodoxin as electron donor, a maximal catalytic activity of 4500 nmol C2H4 formed X min-1 X (mg Fe protein)-1 was measured in vitro for the Fe protein. The results are discussed in relation to the present model for nitrogenase catalysis.

Azotobacter↗

PubMed1985

Properties of the MgATP and MgADP binding sites on the Fe protein of nitrogenase from Azotobacter vinelandii.

Flow dialysis was used to study the binding of MgATP and MgADP to the nitrogenase proteins of Azotobacter vinelandii. Both reduced and oxidized Av2 bind two molecules of MgADP, with the following dissociation constants: reduced Av2, K1 = 0.091 +/- 0.021 mM and K2 = 0.044 +/- 0.009 mM; oxidized Av2, K1 = 0.024 +/- 0.015 mM and K2 = 0.039 +/- 0.022 mM. Binding of MgADP to reduced Av2 shows positive co-operativity. Oxidized Av2 binds two molecules of MgATP with dissociation constants K1 = 0.049 +/- 0.016 mM and K2 = 0.18 +/- 0.05 mM. Binding data of MgATP to reduced Av2 can be fitted by assuming one binding site, but a better fit was obtained by assuming two binding sites on the protein with negative co-operativity and with dissociation constants K1 = 0.22 +/- 0.03 mM and K2 = 1.71 +/- 0.50 mM. It was found that results concerning the number of binding sites and the dissociation constants of MgATP-Av2 and MgADP-Av2 complexes depend to a great extent on the specific activity of the Av2 preparation used, and that it is difficult to correct binding data for inactive protein. No binding of MgADP to Av1 could be demonstrated. Binding studies of MgADP to a mixture of Av1 and Av2 showed that Av1 did not affect the binding of MgADP to either oxidized or reduced Av2. Inhibition studies were performed to investigate the interaction of MgATP and MgADP binding to oxidized and reduced Av2. All the experimental data can be explained by the minimum hypothesis, i.e. the presence of two adenine nucleotide binding sites on Av2. MgATP and MgADP compete for these two binding sites on the Fe protein.

Adenosine Diphosphate↗

PubMed1985

Electron microscopy of the Mo-Fe-protein from Azotobacter vinelandii nitrogenase.

The quaternary structure of the Mo-Fe-protein from Azotobacter vinelandii has been studied by electron microscopy. A model of the molecule of the Mo-Fe-protein has been proposed: two alpha subunits are displaced relative to two beta subunits along a twofold axis, so the molecule can be characterized by the point-group pseudosymmetry 222. Computer averaging of the images showed that one of the projections of the molecule could be characterized by twofold rotational symmetry. Micrographs of nitrogenase recombined complex (Mo-Fe-protein + Fe-protein) have been obtained. They showed particles close in size and form to the Mo-Fe-protein molecule. Therefore, it has been proposed that the Fe-protein could be situated in the central cavity of Mo-Fe-protein.

Azotobacter↗

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↗

PubMed1986

Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii.

The involvement of the cytoplasmic membrane in electron transport to nitrogenase has been studied. Evidence shows that nitrogenase activity in Azotobacter vinelandii is coupled to the flux of electrons through the respiratory chain. To obtain information about proteins involved, the changes occurring in A. vinelandii cells transferred to nitrogen-free medium after growth on NH4Cl (depression of nitrogenase activity) were studied. Synthesis of the nitrogenase polypeptides was detectable 5 min after transfer to nitrogen-free medium. No nitrogenase activity could be detected until t = 20 min, whereupon a linear increase of nitrogenase activity with time was observed. Synthesis of nitrogenase was accompanied by synthesis of flavodoxin II and two membrane-bound polypeptides of Mr 29,000 and 30,000. Analysis with respect to changes in membrane-bound NAD(P)H dehydrogenase activities revealed the induction of an NADPH dehydrogenase activity, which was not detectable in membranes isolated from cells grown in the presence of NH4OAc. This induced activity was associated with the appearance of a polypeptide of Mr 29,000 in the NADPH dehydrogenase complex.

Azotobacter↗

PubMed1986

Structure predictions and surface charge of nitrogenase flavodoxins from Klebsiella pneumoniae and Azotobacter vinelandii.

A first approximation to the tertiary structure of the nitrogenase flavodoxins of Klebsiella pneumoniae and Azotobacter vinelandii can be obtained by superimposing their amino acid sequences upon the crystallographically determined structure of the long-chain flavodoxin from Anacystis nidulans. This procedure is validated by secondary structure predictions based on the sequence alone and by the distribution of polar and hydrophobic residues. It reveals, among other things, a distinctive distribution of surface charge peculiar to the nitrogenase flavodoxins, which is probably important in determining the kinetics of electron transfer with their physiological redox partners. The most likely positions of the phosphodiester bridge which has been described in the A. vinelandii molecule can also be assessed.

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↗

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↗