Crystallization and preliminary X-ray investigation of lipoamide dehydrogenase from Azotobacter vinelandii

The FAD-containing enzyme lipoamide dehydrogenase (EC 1.6.4.3. NADH: lipoamide oxidoreductase) of Azotobacter vinelandii has been crystallized from polyethylene glycol solutions. The space group is P2(1)2(1)2(1) with one dimer in the asymmetric unit. The cell dimensions are: a = 64.2, b = 83.8, c = 193 A. X-ray reflections extend to at least 2.2 A resolution.     

Interaction of lipoamide dehydrogenase with the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

The interaction between lipoamide dehydrogenase (E3) and dihydrolipoyl transacetylase (E2p) from the pyruvate dehydrogenase complex was studied during the reconstitution of monomeric E3 apoenzymes from Azotobacter vinelandii and Pseudomonas fluorescens. The dimeric form of E3 is not only essential for catalysis but also for binding to the E2p core, because the apoenzymes as well as a monomeric holoenzyme from P. fluorescens, which can be stabilized as an intermediate at 0 degree C, do not bind to E2p. Lipoamide dehydrogenase from A. vinelandii contains a C-terminal extension of 15 amino acids with respect to glutathione reductase which is, in contrast to E3, presumably not part of a multienzyme complex. Furthermore, the last 10 amino acid residues of E3 are not visible in the electron density map of the crystal structure and are probably disordered. Therefore, the C-terminal tail of E3 might be an attractive candidate for a binding region. To probe this hypothesis, a set of deletions of this part was prepared by site-directed mutagenesis. Deletion of the last five amino acid residues did not result in significant changes. A further deletion of four amino acid residues resulted in a decrease of lipoamide activity to 5% of wild type, but the binding to E2p was unaffected. Therefore it is concluded that the C-terminus is not directly involved in binding to the E2p core. Deletion of the last 14 amino acids produced an enzyme with a high tendency to dissociate (Kd approximately 2.5 microM). This mutant binds only weakly to E2p. The diaphorase activity was still high. This indicates, together with the decreased Km for NADH, that the structure of the monomer is not appreciably changed by the mutation. Rather the orientation of the monomers with respect to each other is changed. It can be concluded that the binding region of E3 for E2p is constituted from structural parts of both monomers and binding occurs only when dimerization is complete.     

Molecular relaxation spectroscopy of flavin adenine dinucleotide in wild type and mutant lipoamide dehydrogenase from Azotobacter vinelandii.

The temperature dependence of the fluorescence emission spectra of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii shows that the protein matrix in the vicinity of the prosthetic group is rigid on a nanosecond time scale in a medium of high viscosity (80% glycerol). The active site of a deletion mutant of this enzyme, which lacks 14 C-terminal amino acids, is converted from a solid-state environment (on the nanosecond time scale of fluorescence) into a state where efficient dipolar relaxation takes place at temperatures between 203 and 303 K. In aqueous solution, fast dipolar fluctuations are observed in both proteins. It is shown from fluorescence quenching of the flavin by iodide ions that the prosthetic groups of the mutant protein are partially iodide accessible in contrast to the wild type enzyme. A detailed analysis of the temperature dependence of spectral energies according to continuous relaxation models reveals two distinct relaxation processes in the deletion mutant, which were assigned to solvent and protein dipoles, respectively. From the long-wavelength shifts of the emission spectra upon red-edge excitation, it is demonstrated that the active site of the wild type enzyme has high structural homogeneity in comparison to the deletion mutant. In combination with results obtained by X-ray diffraction studies on crystals of the wild type enzyme, it can be concluded that the C-terminal polypeptide of the A. vinelandii enzyme interacts with the dehydrolipoamide binding site, thereby shielding the flavins from the solvent.     

X-ray structure of lipoamide dehydrogenase from Azotobacter vinelandii determined by a combination of molecular and isomorphous replacement techniques

The crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii has been determined by a combination of molecular replacement and isomorphous replacement techniques yielding eventually a good-quality 2.8 A electron density map. Initially, the structure determination was attempted by molecular replacement procedures alone using a model of human glutathione reductase, which has 26% sequence identity with this bacterial dehydrogenase. The rotation function yielded the correct orientation of the model structure both when the glutathione reductase dimer and monomer were used as starting model. The translation function could not be solved, however. Consequently, data for two heavy-atom derivatives were collected using the Hamburg synchotron facilities. The derivatives had several sites in common, which was presumably a major reason why the electron density map obtained by isomorphous information alone was of poor quality. Application of solvent flattening procedures cleaned up the map considerably, however, showing clearly the outline of the lipoamide dehydrogenase dimer, which has a molecular weight of 100,000. Application of the "phased translation function", which combines the phase information of both isomorphous and molecular replacement, led to an unambiguous determination of the position of the model structure in the lipoamide dehydrogenase unit cell. The non-crystallographic 2-fold axis of the dimer was optimized by several cycles of constrained-restrained least-squares refinement and subsequently used for phase improvement by 2-fold density averaging. After ten cycles at 3.5 A, the resolution was gradually extended to 2.8 A in another 140 cycles. The 2.8 A electron density distribution obtained in this manner was of much improved quality and allowed building of an atomic model of A. vinelandii lipoamide dehydrogenase. It appears that in the orthorhombic crystals used each dimer is involved in contacts with eight surrounding dimers, leaving unexplained why the crystals are rather fragile. Contacts between subunits within one dimer, which are quite extensive, can be divided into two regions separated by a cavity. In one of the contact regions, the level of sequence identity with glutathione reductase is very low but it is quite high in the other. The folding of the polypeptide chain in each subunit is quite similar to that of glutathione reductase, as is the extended conformation of the co-enzyme FAD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. A comparison with the structure of glutathione reductase

The structure of lipoamide dehydrogenase from Azotobacter vinelandii has been refined by the molecular dynamics technique to an R-factor of 19.8% at 2.2 A resolution. In the final model, the root-mean-square deviation from ideality is 0.02 A for bond lengths and 3.2 degrees for bond angles. The asymmetric unit comprises two subunits, each consisting of 466 amino acid residues and the prosthetic group FAD, plus 512 solvent molecules. The last ten amino acid residues of both chains are not visible in the electron density distribution and they are probably disordered. The operation required to superimpose the two chains forming the dimer is a rotation of exactly 180 degrees with no translation component. The final model shows the two independently refined subunits to be very similar, except for six loops located at the surface of the molecule. The structure of each subunit of the enzyme consists of four domains with the catalytic centre located at the subunit interface. The reactive disulphide bridge, 48-53, is oxidized with S gamma of Cys53 located 3.5 A away from carbon C-4a of the isoalloxazine ring. The side-chain of His450' points its N epsilon 2 towards S gamma of Cys48 and is hydrogen bonded to the carboxylate of Glu455'. The FAD is bound in an extended conformation and the isoalloxazine ring is not completely planar with an angle between the pteridine and the benzene ring of 7.3 degrees in the first subunit and of 12.1 degrees in the second one. The overall folding of lipoamide dehydrogenase is very similar to that of glutathione reductase. However, a comparison of the two enzymes, which have only 26% sequence identity, reveals significant conformational differences. These concern the tertiary as well as the quaternary structure of the two molecules. In each subunit of lipoamide dehydrogenase the NAD-binding domain and the interface domain appear to be differently oriented with respect to the FAD-binding domain by 7.1 degrees and 7.8 degrees, respectively. The interface domain contains, in addition, major changes in tertiary structure. Furthermore, the two subunits forming the dimer appear to be shifted with respect to each other by more than 4 A, when the lipoamide dehydrogenase dimer is compared with that of glutathione reductase. In spite of all these changes at the tertiary and quaternary level the active sites of the enzymes, which occur at the dimer interface, appear to be remarkably similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pyruvate dehydrogenase from Azotobacter vinelandii. Properties of the N-terminally truncated enzyme.

The pyruvate dehydrogenase multienzyme complex (PDHC) catalyses the oxidative decarboxylation of pyruvate and the subsequent acetylation of coenzyme A to acetyl-CoA. Previously, limited proteolysis experiments indicated that the N-terminal region of the homodimeric pyruvate dehydrogenase (E1p) from Azotobacter vinelandii could be involved in the binding of E1p to the core protein (E2p) [Hengeveld, A. F., Westphal, A. H. & de Kok, A. (1997) Eur J. Biochem. 250, 260-268]. To further investigate this hypothesis N-terminal deletion mutants of the E1p component of Azotobacter vinelandii pyruvate dehydrogenase complex were constructed and characterized. Up to nine N-terminal amino acids could be removed from E1p without effecting the properties of the enzyme. Truncation of up to 48 amino acids did not effect the expression or folding abilities of the enzyme, but the truncated enzymes could no longer interact with E2p. The 48 amino acid deletion mutant (E1pdelta48) is catalytically fully functional: it has a Vmax value identical to that of wild-type E1p, it can reductively acetylate the lipoamide group attached to the lipoyl domain of the core enzyme (E2p) and it forms a dimeric molecule. In contrast, the S0.5 for pyruvate is decreased. A heterodimer was constructed containing one subunit of wild-type E1p and one subunit of E1pdelta48. From the observation that the heterodimer was not able to bind to E2p, it is concluded that both N-terminal domains are needed for the binding of E1p to E2p. The interactions are thought to be mainly of an electrostatic nature involving negatively charged residues on the N-terminal domains of E1p and previously identified positively charged residues on the binding and catalytic domain of E2p.     

Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study.

Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K. The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme. The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis. The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes. Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme. Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins. The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system. By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated. For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A. vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered. In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved. The anisotropy decay parameters also provide information on electronic and dipolar relaxational properties of the flavin active site. The local environment of the prosthetic groups in the deletion mutant of the A. vinelandii enzyme is highly inhomogeneous, and a transition from slow to rapid dipolar relaxation is observed over the measured temperature range. In the highly homogeneous active site of the wild-type enzyme, dipolar relaxation is slowed down beyond the time scale of fluorescence emission at any temperature studied. Our results are in favor of a COOH-terminal polypeptide interacting with the active site, thereby shielding the isoalloxazines from the solvent. This biological system forms a very appropriate tool to test the validity of photophysical models describing homoenergy transfer.     

The pyruvate-dehydrogenase complex from Azotobacter vinelandii.

The pyruvate dehydrogenase complex from Axotobacter vinelandii was isolated in a five-step procedure. The minimum molecular weight of the pure complex is 600,000, as based on an FAD content of 1.6 nmol-mg protein-1. The molecular weight is 1.0-1.2 X 10(6), indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr89,000) and lipoamide dehydrogenase (Mrmonomer 56,000) two active transacetylase isoenzymes are present with molecular weight on the gel 82,000 and 59,000 but probably actually lower. The pure complex has a specific activity of the pyruvate-NAD+ reductase (overall) reaction of 10 units-mg protein-1 at 25 degrees C. The partial reactions have the following specific activities in units-mg protein-1 at 25 degrees C under standard conditions: pyruvate-K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor. Under aerobic conditios pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 mumol 02-min-1-mg-1, Km(pyruvate)0.3 mM. The kinetics of this reaction shows a linear 1/velocity-1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity is stimulated by AMP and sulphate and is inhibited by acetyl-CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.     

A preliminary crystallographic study of isocitrate dehydrogenase from Azotobacter vinelandii

Large single crystals of isocitrate dehydrogenase from Azotobacter vinelandii have been grown by vapor diffusion from ammonium sulfate and phosphate solutions. The crystals are tetragonal, space group P4₂2₁2 with cell dimensions $\text{a = 122.1}\text{A}\text{?}$, $\text{c = 163.9}\text{a}\text{?}$. There are two molecules of 80,000 molecular weight per asymmetric unit. Native data to 5.5 Å resolution have been collected on a diffractometer. A rotation function using data between 10 Å and 6 Å resolution indicates three possible orientations of the non-crystallographic 2-fold axis relating the two molecules.     

Fluorescence energy-transfer studies on the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii.

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (greater than or equal to 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis. Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.     

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.     

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.     

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.