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Query: UNIPROT:P06889 (Mol)
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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)
J Mol Biol 1991 Aug 20
PMID:Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. A comparison with the structure of glutathione reductase. 188 Aug 7

The role of protein residues in activating the substrate in the reaction catalyzed by the flavoprotein p-hydroxybenzoate hydroxylase was studied. X-ray crystallography (Schreuder, H. A., Prick, P.A.J., Wieringa, R.K., Vriend, G., Wilson, K.S., Hol, W.G. J., and Drenth, J. (1989) J. Mol. Biol. 208, 679-696) indicates that Tyr-201 and Tyr-385 form a hydrogen bond network with the 4-OH of p-hydroxybenzoate. Therefore, site directed mutants were constructed, converting each of these tyrosines into phenylalanines. Spectral (visible and fluorescence) properties, reduction potentials, and binding constants are very similar to those of wild type, indicating that there are no major structural changes in the mutants. In the absence of substrate, the mutants and wild type exhibit similar pH-dependent changes in the FAD spectrum. However, the enzyme-substrate complex of Tyr-201----Phe lacks an ionization observed in both wild type and Tyr-385----Phe, which preferentially bind the phenolate form of substrates. Tyr-201----Phe shows no preference, indicating that Tyr-201 is required to ionize the substrate. The mutants have less than 6% the activity of the wild type enzyme. The effects on catalysis were studied by stopped flow techniques. Reduction of FAD by NADPH is slower by 10-fold in Tyr-201----Phe and 100-fold in Tyr-385----Phe. When the reduced Tyr-201----Phe-p-hydroxybenzoate complex reacts with oxygen, a long-lived flavin-C(4a)-hydroperoxide is observed, which slowly eliminates H2O2 with very little hydroxylation. Thus, the role of Tyr-201 is to activate the substrate by stabilizing the phenolate. Tyr-385----Phe reacts with oxygen to form 25% oxidized enzyme, and 75% flavin hydroperoxide, which successfully hydroxylates the substrate. This mutant also hydroxylates the product (3, 4-dihydroxybenzoate) to form gallic acid.
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PMID:Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants. 191 43

The crystal structure of NADH peroxidase (EC 1.11.1.1) from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been refined to a resolution of 2.16 A using the simulated annealing method. The final crystallographic R-factor is 17.7% for all data in the resolution range 7 to 2.16 A. The standard deviations are 0.015 A in bond lengths and 3.0 degrees in bond angles for the final model, which includes all 447 amino acid residues, one FAD and 369 water molecules. The enzyme is a symmetrical tetramer with point group D2; the symmetry is crystallographic. The redox center of the enzyme consists of FAD and a cysteine (Cys42), which forms a sulfenic acid (Cys-SOH) in its oxidized state. A histidine (His10) close to Cys42 is likely to act as an active-site base. In the analyzed crystal, the enzyme was in a non-native oxidation state with Cys42 oxidized to a sulfonic acid Cys-SO3H. The chain fold of NADH peroxidase is similar to those of disulfide oxidoreductases. A comparison with glutathione reductase, a representative of this enzyme family, is given.
J Mol Biol 1991 Oct 20
PMID:Structure of NADH peroxidase from Streptococcus faecalis 10C1 refined at 2.16 A resolution. 194 54

Cholesterol oxidase (3 beta-hydroxysteroid oxidase, EC 1.1.3.6) is an FAD-dependent enzyme that carries out the oxidation and isomerization of steroids with a trans A : B ring junction. The crystal structure of the enzyme from Brevibacterium sterolicum has been determined using the method of isomorphous replacement and refined to 1.8 A resolution. The refined model includes 492 amino acid residues, the FAD prosthetic group and 453 solvent molecules. The crystallographic R-factor is 15.3% for all reflections between 10.0 A and 1.8 A resolution. The structure is made up of two domains: an FAD-binding domain and a steroid-binding domain. The FAD-binding domain consists of three non-continuous segments of sequence, including both the N terminus and the C terminus, and is made up of a six-stranded beta-sheet sandwiched between a four-stranded beta-sheet and three alpha-helices. The overall topology of this domain is very similar to other FAD-binding proteins. The steroid-binding domain consists of two non-continuous segments of sequence and contains a six-stranded antiparallel beta-sheet forming the "roof" of the active-site cavity. This large beta-sheet structure and the connections between the strands are topologically similar to the substrate-binding domain of the FAD-binding protein para-hydroxybenzoate hydroxylase. The active site lies at the interface of the two domains, in a large cavity filled with a well-ordered lattice of 13 solvent molecules. The flavin ring system of FAD lies on the "floor" of the cavity with N-5 of the ring system exposed. The ring system is twisted from a planar conformation by an angle of approximately 17 degrees, allowing hydrogen-bond interactions between the protein and the pyrimidine ring of FAD. The amino acid residues that line the active site are predominantly hydrophobic along the side of the cavity nearest the benzene ring of the flavin ring system, and are more hydrophilic on the opposite side near the pyrimidine ring. The cavity is buried inside the protein molecule, but three hydrophobic loops at the surface of the molecule show relatively high temperature factors, suggesting a flexible region that may form a possible path by which the substrate could enter the cavity. The active-site cavity contains one charged residue, Glu361, for which the side-chain electron density suggests a high degree of mobility for the side-chain. This residue is appropriately positioned to act as the proton acceptor in the proposed mechanism for the isomerization step.
J Mol Biol 1991 Jun 05
PMID:Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A resolution. 205 87

Using specific monooxygenase and oxidase inhibitors in a plant cell/microbe coincubation assay, the biochemical mechanisms of the plant activation of two aromatic amines were compared. The biological endpoints included mutation induction, inhibition of mutagenicity, viability of the plant cells (activating system), and viability of the microbial cells (genetic indicator organism). The activation of m-phenylenediamine by TX1 cells was mediated by enzyme systems that were inhibited by diethyldithiocarbamate, potassium cyanide, methimazole, (+)-catechin or acetaminophen. The inhibition by metyrapone was attended by toxicity in the plant cells. These data implicate a TX1 cell peroxidase and a FAD-dependent monooxygenase in the plant activation of m-phenylenediamine. The TX1 cell activation of 2-aminofluorene was inhibited by diethyldithiocarbamate, 7,8-benzoflavone, acetaminophen or (+)-catechin. An additional pathway of the plant cells in the activation of 2-aminofluorene may involve a cytochrome P-448-type N-hydroxylase.
Environ Mol Mutagen 1990
PMID:The biochemical mechanisms of the plant activation of promutagenic aromatic amines. 216 71

The oxidation of alkanes to alkanols by Pseudomonas oleovorans involves a three-component enzyme system: alkane hydroxylase, rubredoxin and rubredoxin reductase. Alkane hydroxylase and rubredoxin are encoded by the alkBFGHJKL operon, while previous studies indicated that rubredoxin reductase is most likely encoded on the second alk cluster: the alkST operon. In this study we show that alkT encodes the 41 x 10(3) Mr rubredoxin reductase, on the basis of a comparison of the expected amino acid composition of AlkT and the previously established amino acid composition of the purified rubredoxin reductase. The alkT sequence revealed significant similarities between AlkT and several NAD(P)H and FAD-containing reductases and dehydrogenases. All of these enzymes contain two ADP binding sites, which can be recognized by a common beta alpha beta-fold or fingerprint, derived from known structures of cofactor binding enzymes. By means of this amino acid fingerprint we were able to determine that one ADP binding site in rubredoxin reductase (AlkT) is located at the N terminus and is involved in FAD binding, while the second site is located in the middle of the sequence and is involved in the binding of NAD or NADP. In addition, we derived from the sequences of FAD binding reductases a second amino acid fingerprint for FAD binding, and we used this fingerprint to identify a third amino acid sequence in AlkT near the carboxy terminus for binding of the flavin moiety of FAD. On the basis of the known architecture and relative spatial orientations of the NAD and FAD binding sites in related dehydrogenases, a model for part of the tertiary structure of AlkT was developed.
J Mol Biol 1990 Mar 05
PMID:Rubredoxin reductase of Pseudomonas oleovorans. Structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints. 231 93

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1989 Aug 20
PMID:Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes. 255 83

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)
J Mol Biol 1989 Mar 20
PMID:X-ray structure of lipoamide dehydrogenase from Azotobacter vinelandii determined by a combination of molecular and isomorphous replacement techniques. 271 52

The two structural genes encoding tobacco nitrate reductases (NR) were isolated from tobacco genomic libraries constructed in lambda EMBL phages. Two independent genomic clones of 12.6 and 13.5 kbp, respectively, cross-hybridizing with a partial tobacco NR cDNA probe, were further characterized. Southern blot experiments were performed with the NR cDNA probe on genomic DNA derived from Nicotiana tabacum and from the ancestors of tobacco, N. sylvestris and N. tomentosiformis. They showed that the larger clone, referred to as nia-1, was related to the N. tomentosiformis parent, and the smaller one, referred to as nia-2, to the N. sylvestris parent. Both homeologous genes were found to be expressed in tobacco. The sequence of the gene nia-2, from which the cDNA previously cloned is derived, was determined. It encodes a 904 amino acid protein. Three intervening sequences were found interspersed with the coding sequence of the enzyme. The precise location of the transcription initiation site on the structural gene was mapped by primer extension experiments. A TATA consensus sequence was detected 32 bp upstream from the transcription initiation site. The leader sequence of the transcript is 138 nucleotides long and a stable secondary structure involving the translation initiation site has been proposed. The amino acid sequence of tobacco NR deduced from the nucleotide sequence of the gene shows that heme and FAD binding domains occupy the entire C-terminal moiety of the polypeptide. The remaining N-terminal part of the protein should thus carry the catalytic site of nitrate reduction by the molybdenum cofactor.
Mol Gen Genet 1989 Mar
PMID:Molecular cloning and characterisation of the two homologous genes coding for nitrate reductase in tobacco. 273 90

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.
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PMID:Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene. 283 61


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