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V Biou

Publications and source records attributed to V Biou.

18 recordsLinked to original sources
PubMed2001

MAD on threonine synthase: the phasing power of oxidized selenomethionine.

The use of selenomethionine and anomalous dispersion has become the most widely used way of solving the phase problem for de novo protein structure determination. In this paper, MAD data collected from oxidized and reduced selenomethionine-containing protein are described, and it is shown that oxidized selenomethionine has a very strong phasing power and can be efficiently used if the oxidation is uniform. The comparison was performed on threonine synthase crystals. For example, the phasing power of the oxidized data is doubled for the dispersive signal and is 20% stronger for the anomalous signal at the peak wavelength. The strength of the anomalous signal can be used to improve the signal when a protein contains few methionines or for single anomalous dispersion. The oxidation of some selenomethionines shows in the electron-density map through the presence of water molecules within hydrogen-bonding distance of the putative O atom.

Arabidopsis↗
PubMed2001

Enzymology, structure, and dynamics of acetohydroxy acid isomeroreductase.

Acetohydroxy acid isomeroreductase is a key enzyme involved in the biosynthetic pathway of the amino acids isoleucine, valine, and leucine. This enzyme is of great interest in agrochemical research because it is present only in plants and microorganisms, making it a potential target for specific herbicides and fungicides. Moreover, it catalyzes an unusual two-step reaction that is of great fundamental interest. With a view to characterizing both the mechanism of inhibition by potential herbicides and the complex reaction mechanism, various techniques of enzymology, molecular biology, mass spectrometry, X-ray crystallography, and theoretical simulation have been used. The results and conclusions of these studies are described briefly in this paper.

Alcohol Oxidoreductases↗
PubMed2001

Crystal structure of threonine synthase from Arabidopsis thaliana.

Threonine synthase (TS) is a PLP-dependent enzyme that catalyzes the last reaction in the synthesis of threonine from aspartate. In plants, the methionine pathway shares the same substrate, O-phospho-L-homoserine (OPH), and TS is activated by S-adenosyl-methionine (SAM), a downstream product of methionine synthesis. This positive allosteric effect triggered by the product of another pathway is specific to plants. The crystal structure of Arabidopsis thaliana apo threonine synthase was solved at 2.25 A resolution from triclinic crystals using MAD data from the selenomethionated protein. The structure reveals a four-domain dimer with a two-stranded beta-sheet arm protruding from one monomer onto the other. This domain swap could form a lever through which the allosteric effect is transmitted. The N-terminal domain (domain 1) has a unique fold and is partially disordered, whereas the structural core (domains 2 and 3) shares the functional domain of PLP enzymes of the same family. It also has similarities with SAM-dependent methyltransferases. Structure comparisons allowed us to propose potential sites for pyridoxal-phosphate and SAM binding on TS; they are close to regions that are disordered in the absence of these molecules.

Amino Acid Sequence↗
PubMed2000

Structure of spinach acetohydroxyacid isomeroreductase complexed with its reaction product dihydroxymethylvalerate, manganese and (phospho)-ADP-ribose.

Acetohydroxyacid isomeroreductase catalyses a two-step reaction composed of an alkyl migration followed by an NADPH-dependent reduction. Both steps require a divalent cation and the first step has a strong preference for magnesium. Manganese ions are highly unfavourable to the reaction: only 3% residual activity is observed in the presence of this cation. Acetohydroxyacid isomeroreductase has been crystallized with its substrate, 2-aceto-2-hydroxybutyrate (AHB), Mn(2+) and NADPH. The 1.6 A resolution electron-density map showed the reaction product (2,3-dihydroxy-3-methylvalerate, DHMV) and a density corresponding to (phospho)-ADP-ribose instead of the whole NADP(+). This is one of the few structures of an enzyme complexed with its reaction product. The structure of this complex was refined to an R factor of 19.3% and an R(free) of 22.5%. The overall structure of the enzyme is very similar to that of the complex with the reaction-intermediate analogue IpOHA [N-hydroxy-N-isopropyloxamate; Biou et al. (1997), EMBO J. 16, 3405-3415]. However, the active site shows some differences: the nicotinamide is cleaved and the surrounding amino acids have rearranged accordingly. Comparison between the structures corresponding to the reaction intermediate and to the end of the reaction allowed the proposal of a reaction scheme. Taking this result into account, the enzyme was crystallized with Ni(2+) and Zn(2+), for which only 0.02% residual activity were measured; however, the crystals of AHB/Zn/NADPH and of AHB/Ni/NADPH also contain the reaction product. Moreover, mass-spectrometry measurements confirmed the -cleavage of nicotinamide.

Adenosine Diphosphate Ribose↗
PubMed1999

Characterization of the conformational changes of acetohydroxy acid isomeroreductase induced by the binding of Mg2+ ions, NADPH, and a competitive inhibitor.

Acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the parallel branched chain amino acid pathway, is a homodimer with an Mr of approximately 114000 which in the presence of Mg2+ ions catalyzes an unusual alkyl migration followed by an NADPH-dependent reduction. Prior binding of NADPH and Mg2+ to the enzyme was shown to be required for substrate or competitive inhibitor [N-hydroxy-N-isopropyloxamate (IpOHA)] binding [Dumas, R., et al. (1994) Biochem. J. 301, 813-820]. Moreover, crystallographic data for the enzyme-NADPH-Mg2+-IpOHA complex [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] have shown that IpOHA was completely buried inside the active site. These observations raised the question of how the reaction intermediate analogue inhibitor can reach the active site and implied that conformational changes occurred during the binding process. With a view of characterizing these conformational changes, H-D exchange experiments combined with mass spectrometry were performed. Results demonstrated that Mg2+ ions and NADPH binding led to an initial conformational change at the interface of the two domains of each monomer. Binding of the two cofactors to isomeroreductase alters the structure of the active site to promote inhibitor (substrate) binding, in agreement with the ordered mechanism of the enzyme. Structural changes remote from the active site were also found. They were interpreted as long-range structural effects on the two domains and on the two monomers in the time course of the ligand binding process.

Alcohol Oxidoreductases↗
PubMed1998

A loop deletion in the plant acetohydroxy acid isomeroreductase homodimer generates an active monomer with reduced stability and altered magnesium affinity.

Plant acetohydroxy acid isomeroreductase is a stable homodimer which catalyzes in the presence of magnesium an alkyl migration followed by a NADPH-dependent reduction. Since the enzyme exhibits no kinetic cooperativity either for its cofactor (NADPH and magnesium) or for its substrates, the reason for dimerization of this enzyme was not obvious. Recently, crystallographic studies [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] revealed that the loop of residues 422-431 plays a major part in the dimer interface. To understand the role of the quaternary structure of the enzyme, we have deleted residues 423-430 and substituted Phe 431 for serine. This mutant was further overproduced in Escherichia coli, purified to homogeneity, and characterized. Gel filtration and thermodynamic experiments disclosed that this mutant behaves as an active monomer with reduced thermal stability. Furthermore, kinetic and fluorescence experiments showed that the behavior of the monomer with respect to magnesium was greatly altered. These results demonstrate the function of the quaternary structure of plant acetohydroxy acid isomeroreductase in the stabilization of the tertiary structure but also in the stabilization of a high-affinity magnesium binding site.

Alcohol Oxidoreductases↗
PubMed1998

Kinetic and mass spectrometric analyses of the interactions between plant acetohydroxy acid isomeroreductase and thiadiazole derivatives.

Plant acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the branched chain amino acid biosynthetic pathway, has been submitted to high-throughput screening for herbicide discovery. We report here the discovery of a new class of compounds belonging to the thiadiazole family, which exhibit a strong inhibitory effect on this plant enzyme. Kinetic analyses revealed that these compounds act as either reversible or irreversible noncompetitive inhibitors of the plant enzyme. Reversibility or irreversibility of these compounds can be attributed to the nature of the additional groups of the thiadiazole ring favoring or not favoring the formation of a covalent adduct. Mass spectrometric experiments on the complex between an irreversible compound belonging to the thiadiazole family and the plant enzyme identified Cys498 as the binding site of the inhibitor.

Alcohol Oxidoreductases↗
PubMed1998

Three-dimensional visualization of the distribution, growth, and regeneration of monoaminergic neurons in whole mounts of immature mammalian CNS.

At birth, the opossum, Monodelphis domestica, corresponds roughly to a 14-day-old mouse embryo. The aim of these experiments was to compare the distribution of monoaminergic neurons in the two preparations during development and to follow their regeneration after injury. Procedures that allowed antibody staining to be visible in transparent whole mounts of the entire central nervous system (CNS) were devised. Neurons throughout the brain and spinal cord were stained for tyrosine hydroxylase (TH) and for serotonin (5-HT). At birth, patterns of monoaminergic cells in opossum CNS resembled those found in 14-day mouse embryos and other eutherian mammals. By postnatal day 5, immunoreactive cell bodies were clustered in appropriate regions of the midbrain and hindbrain, and numerous axons were already present throughout the spinal cord. Differences found in the opossum were the earlier presence of TH neurons in the olfactory bulb and of 5-HT neuronal perikarya in the spinal cord. Most, if not all, monoaminergic neurons in opossum were already postmitotic at birth. To study regeneration, crushes were made in cervical cords in culture. By 5 days, 8% of all TH-labeled axons and 14% of serotonergic axons had grown beyond lesions. Distal segments of monoaminergic axons degenerated. In CNS preparations from opossums older than 11 days, no regeneration of monoaminergic fibers occurred. Isolated embryonic mouse CNS also showed regeneration across spinal cord lesions, providing the possibility of using knockout and transgenic animals. Our procedures for whole-mount observation of identified cell bodies and their axons obviates the need for serial reconstructions and allows direct comparison of events occurring during development and regeneration.

Animals↗
PubMed1997

The crystal structure of dimeric kinesin and implications for microtubule-dependent motility.

The dimeric form of the kinesin motor and neck domain from rat brain with bound ADP has been solved by X-ray crystallography. The two heads of the dimer are connected via a coiled-coil alpha-helical interaction of their necks. They are broadly similar to one another; differences are most apparent in the head-neck junction and in a moderate reorientation of the neck helices in order to adopt to the coiled-coil conformation. The heads show a rotational symmetry (approximately 120 degrees) about an axis close to that of the coiled-coil. This arrangement is unexpected since it is not compatible with the microtubule lattice. In this arrangement, the two heads of a kinesin dimer could not have equivalent interactions with microtubules.

Adenosine Diphosphate↗
PubMed1997

The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution.

Acetohydroxy acid isomeroreductase catalyzes the conversion of acetohydroxy acids into dihydroxy valerates. This reaction is the second in the synthetic pathway of the essential branched side chain amino acids valine and isoleucine. Because this pathway is absent from animals, the enzymes involved in it are good targets for a systematic search for herbicides. The crystal structure of acetohydroxy acid isomeroreductase complexed with cofactor NADPH, Mg2+ ions and a competitive inhibitor with herbicidal activity, N-hydroxy-N-isopropyloxamate, was solved to 1.65 A resolution and refined to an R factor of 18.7% and an R free of 22.9%. The asymmetric unit shows two functional dimers related by non-crystallographic symmetry. The active site, nested at the interface between the NADPH-binding domain and the all-helical C-terminus domain, shows a situation analogous to the transition state. It contains two Mg2+ ions interacting with the inhibitor molecule and bridged by the carboxylate moiety of an aspartate residue. The inhibitor-binding site is well adjusted to it, with a hydrophobic pocket and a polar region. Only 24 amino acids are conserved among known acetohydroxy acid isomeroreductase sequences and all of these are located around the active site. Finally, a 140 amino acid region, present in plants but absent from other species, was found to make up most of the dimerization domain.

Alcohol Oxidoreductases↗
PubMed1997

Purification and characterization of a fusion protein of plant acetohydroxy acid synthase and acetohydroxy acid isomeroreductase.

The nucleotide sequence coding for the Arabidopsis thaliana acetohydroxy acid synthase was genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in Escherichia coli. This construction allowed the production of large amounts of soluble fusion protein. The pure chimeric enzyme exhibits high acetohydroxy acid synthase and acetohydroxy acid isomeroreductase specific activities. Fusion and native enzymes exhibit similar Km values for their substrates and for most cofactors. Furthermore, whereas native plant acetohydroxy acid synthase is highly unstable, the stability of this enzyme in the fusion has been increased. Thus, the chimeric enzyme appears to be a useful tool for the determination of kinetic and structural properties of plant acetohydroxy acid synthase.

2-Acetolactate Mutase↗
PubMed1995

X-ray crystallography shows that translational initiation factor IF3 consists of two compact alpha/beta domains linked by an alpha-helix.

The structures of the two domains of translational initiation factor IF3 from Bacillus stearothermophilus have been solved by X-ray crystallography using single wavelength anomalous scattering and multiwavelength anomalous diffraction. Each of the two domains has an alpha/beta topology, with an exposed beta-sheet that is reminiscent of several ribosomal and other RNA binding proteins. An alpha-helix that protrudes out from the body of the N-terminal domain towards the C-terminal domain suggests that IF3 consists of two RNA binding domains connected by an alpha-helix and that it may bridge two regions of the ribosome. This represents the first high resolution structural information on a translational initiation factor.

Amino Acid Sequence↗
PubMed1995

Prokaryotic translation initiation factor IF3 is an elongated protein consisting of two crystallizable domains.

We show that translation initiation factor IF3 can be split into two fragments of nearly equal size by the Escherichia coli outer membrane protease omptin. Circular dichroism and small-angle neutron scattering show that the two fragments are structured as domains. Each domain is relatively compact, and they are separated by about 45 A in intact IF3. Thus IF3 is an elongated protein that consists of two well-separated domains. We suggest that these two domains are involved in ribosome binding across the cleft of the 30S ribosome. We also report the crystallization of each domain of IF3.

Circular Dichroism↗
PubMed1994

The 2.9 A crystal structure of T. thermophilus seryl-tRNA synthetase complexed with tRNA(Ser).

The crystal structure of Thermus thermophilus seryl-transfer RNA synthetase, a class 2 aminoacyl-tRNA synthetase, complexed with a single tRNA(Ser) molecule was solved at 2.9 A resolution. The structure revealed how insertion of conserved base G20b from the D loop into the core of the tRNA determines the orientation of the long variable arm, which is a characteristic feature of most serine specific tRNAs. On tRNA binding, the antiparallel coiled-coil domain of one subunit of the synthetase makes contacts with the variable arm and T psi C loop of the tRNA and directs the acceptor stem of the tRNA into the active site of the other subunit. Specificity depends principally on recognition of the shape of tRNA(Ser) through backbone contacts and secondarily on sequence specific interactions.

Adenosine Triphosphate↗
PubMed1992

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.

Crystallization↗
PubMed1990

Secondary structure prediction and protein design.

For non-homologous proteins, and after cross-validation, the methods reviewed in this article exhibit a probability index (percentage of correctly predicted residues per predicted residues) of 59-65.5% according to the methods employed with a standard deviation of 7% for three conformational states: alpha-helix, beta-strand and coil. These present limitations in the accuracy of prediction are related both to the limited number of known structures and to the effect of long-range interactions. The methods based on sequence similarity can improve the accuracy of prediction by expressing the homology of the protein to be predicted explicitly with proteins in the database. Under these circumstances, the probability index can reach 87% with a standard deviation of 6.6%. This property was used for modelling homologous proteins by assisting in amino acid sequence alignments. Examples will be given for the alignment of serine proteinases.

Amino Acid Sequence↗
PubMed1988

Secondary structure prediction: combination of three different methods.

A combination of three complementary secondary structure prediction methods is presented. The methods used are the GOR III method, the Homologue method and a new method, the bit pattern method, which is based on hydrophilic/hydrophobic residue patterns. For this purpose a hydropathy scale was developed and is presented here. The combination algorithm (Combine method) was designed to take the best results of each method and use their differences in order to improve the prediction. The combination yields 65.5% correctly predicted residues in three states: alpha-helix (H), beta-strand (E) and aperiodic structure (C) which is an improvement ranging from 2.5 to 6.5% compared with the individual methods when tested with a 67-polypeptide chain database. Seventy-five per cent of the regular secondary structure (H and E) runs are correctly located and beta-sheet runs are much better located by the Combine method in comparison to the other methods.

Predictive Value of Tests↗