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Genome analyses and the resolution of three–dimensional structures have provided evidence in recent years for hitherto unexpected family relationships between redox proteins of very diverse enzymes involved in bioenergetic electron transport. Many of these enzymes appear in fact to be constructed from only a limited set of building blocks. Phylogenetic analysis of selected units from this ‘redox enzyme construction kit’ indicates an origin for several prominent bioenergetic enzymes that is very early, lying before the divergence of Bacteria and Archaea. Possible scenarios for the early evolution of selected complexes are proposed based on the obtained tree topologies.

The transacting transcriptional activator (Tat) is a viral protein essential for activation of the human immunodeficiency virus (HIV) genes, and it plays an important role in HIV induced immunodeficiency. We report the NMR structural characterization of the active Tat Mal variant that belongs to a highly virulent D-subtype HIV type-1 (HIV-1) strain (Mal) found mainly in Africa. A full Tat Mal protein (87 residues) is synthesized. This synthetic protein is active in a transactivation assay with HeLa cells infected with the HIV long terminal repeated noncoding sequences of the HIV-1 provirus (LTR) lac Z gene. Homonuclear (1)H-NMR spectra allows the sequential assignment of the Tat Mal spin systems. Simulating annealing generates 20 conformers with similar folding. The geometry of the mean structure is optimized with energy minimization to obtain a final structure. As the European variant (Tat Bru) the N-terminal region of Tat Mal constitutes the core, and there is a hydrophobic pocket composed of the conserved Trp 11 interacting with several aromatic residues. The two functional regions of Tat (basic and the cysteine-rich regions) are well exposed to the solvent. A short alpha-helix is observed in region V adjacent to the basic region. This alpha helix induces local structural variations compared to the NMR structure of Tat Bru, and it brings the cysteine-rich and basic regions closer. This study suggests that similar folding exists among Tat variants.

The oxidized and reduced forms of the [Fe4-S4] ferredoxin I from Desulfovibrio desulfuricans Norway were investigated by 1H NMR spectroscopy with the aim of obtaining the complete assignment of the cysteines ligating the cluster. A combination of TOCSY and NOESY measurements together with information from the x-ray crystallographic structure of related ferredoxins provided the sequence-specific assignment of the four cysteines coordinated to the cluster. Through EXSY experiments, the hyperfine shifted resonance signals in the reduced ferredoxin were also assigned. The temperature dependence of the contact-shifted cysteinyl residues of the reduced ferredoxin reveals that two cysteines exhibit anti-Curie behavior whereas the other two cysteines display Curie behavior; that identifies Cys 9 (I) and Cys 15 (III) as ligated to the mixed-valence iron ions.

Rieske/cytochrome b (Rieske/cytb) complexes are proton pumping quinol oxidases that are present in most bacteria and Archaea. The phylogeny of their subunits follows closely the 16S-rRNA phylogeny, indicating that chemiosmotic coupling was already present in the last universal common ancestor of Archaea and bacteria. Haloarchaea are the only organisms found so far that acquired Rieske/cytb complexes via interdomain lateral gene transfer. They encode two Rieske/cytb complexes in their genomes; one of them is found in genetic context with nitrate reductase genes and has its closest relatives among Actinobacteria and the Thermus/Deinococcus group. It is likely to function in nitrate respiration. The second Rieske/cytb complex of Haloarchaea features a split cytochrome b sequence as do Cyanobacteria, chloroplasts, Heliobacteria, and Bacilli. It seems that Haloarchaea acquired this complex from an ancestor of the above-mentioned phyla. Its involvement in the bioenergetic reaction chains of Haloarchaea is unknown. We present arguments in favor of the hypothesis that the ancestor of Haloarchaea, which relied on a highly specialized bioenergetic metabolism, that is, methanogenesis, and was devoid of quinones and most enzymes of anaerobic or aerobic bioenergetic reaction chains, integrated laterally transferred genes into its genome to respond to a change in environmental conditions that made methanogenesis unfavorable.

Antipain [(1-carboxy-2-phenylethyl)carbamoyl-L-arginyl-L-valyl-DL-arginal] is a potent inhibitor of papain and related proteases. The assignment of 1 H NMR signals of the interconverting forms in equilibrium in aqueous solution at DH 5.8 were investigated using two-dimensional DQF-COSY, DOUBLE-RELAYED-COSY, HOHAHA and ROESY nuclear magnetic resonance techniques. The assignments of protons of several forms in equilibrium of the D- and L-arginal antipain isomers were determined in aqueous solution at 300 K, and their corresponding stereochemistry is tentatively proposed. Addition of equimolar amounts of papain resulted in spectral changes and chemical shifts which are compatible with enzyme-L-carbinolamine interactions

The assignments of the proton NMR signals and the conformational analysis of the cystatin-mimicking peptide K13CK (Lys-Val-Gly-Gly-Gln-Val-Val-Cys-Gly-Ala-Pro-Trp-Lys) in aqueous solution at pH 3.2 have been achieved using two-dimensional DQF-COSY, HOHAHA and ROESY nuclear magnetic resonance techniques. The chemical shifts, the magnitudes of the coupling constants, the temperature dependences of the amide protons and the intramolecular NOEs were used to obtain information on the conformation. The NMR spectra of cysteine proteinase inhibitor K13CK demonstrate a cis-trans isomerism at the Ala10Pro11 peptide bond.

Previously published phylogenetic trees reconstructed on "Rieske protein" sequences frequently are at odds with each other, with those of other subunits of the parent enzymes and with small-subunit rRNA trees. These differences are shown to be at least partially if not completely due to problems in the reconstruction procedures. A major source of erroneous Rieske protein trees lies in the presence of a large, poorly conserved domain prone to accommodate very long insertions in well-defined structural hot spots substantially hampering multiple alignments. The remaining smaller domain, in contrast, is too conserved to allow distant phylogenies to be deduced with sufficient confidence. Three-dimensional structures of representatives from this protein family are now available from phylogenetically distant species and from diverse enzymes. Multiple alignments can thus be refined on the basis of these structures. We show that structurally guided alignments of Rieske proteins from Rieske-cytochrome b complexes and arsenite oxidases strongly reduce conflicts between resulting trees and those obtained on their companion enzyme subunits. Further problems encountered during this work, mainly consisting in database errors such as wrong annotations and frameshifts, are described. The obtained results are discussed against the background of hypotheses stipulating pervasive lateral gene transfer in prokaryotes.

Cytochrome c 1 from mitochondrial complex III and the di-heme cytochromes c in the corresponding enzyme from ε-proteobacteria have so far been considered to represent unrelated cytochromes. A missing link protein discovered in the genome of the hyperthermophilic bacterium Aquifex aeolicus , however, provides evidence for a close evolutionary relationship between these two cytochromes. The mono-heme cytochrome c 1 from A. aeolicus contains stretches of strong sequence homology toward the ε-proteobacterial di-heme cytochromes. These di-heme cytochromes are shown to belong to the cytochrome c 4 family. Mapping cytochrome c 1 onto the di-heme sequences and structures demonstrates that cytochrome c 1 results from a mutation-induced collapse of the di-heme cytochrome structure and provides an explanation for its uncommon structural features. The appearance of cytochrome c 1 thus represents an extension of the biological protein repertoire quite different from the widespread innovation by gene duplication and subsequent diversification.

Operons coding for the enzyme arsenite oxidase have been detected in the genomes from Archaea and Bacteria by Blast searches using the amino acid sequences of the respective enzyme characterized in two different beta-proteobacteria as templates. Sequence analyses show that in all these species, arsenite oxidase is transported over the cytoplasmic membrane via the tat system and most probably remains membrane attached by an N-terminal transmembrane helix of the Rieske subunit. The biochemical and biophysical data obtained for arsenite oxidase in the green filamentous bacterium Chloroflexus aurantiacus allow a structural model of the enzyme's membrane association to be proposed. Phylogenies for the two constituent subunits (i.e., the molybdopterin-containing and the Rieske subunit) of the heterodimeric enzyme and their respective homologs in DMSO-reductase, formate dehydrogenase, nitrate reductase, and the Rieske/cytb complexes were calculated from multiple sequence alignments. The obtained phylogenetic trees indicate an early origin of arsenite oxidase before the divergence of Archaea and Bacteria. Evolutionary implications of these phylogenies are discussed.

Primary structures, functional characteristics and phylogenetic relationships of subunits of cytochrome bc complexes from phylogenetically diverse bacterial and archaeal species were analysed. A single case of lateral gene transfer, i.e. the import of an epsilon-proteobacterial cytochrome bc(1) complex into Aquificales, was identified. For the enzyme in the remainder of the species studied, the obtained phylogenies were globally in line with small subunit rRNA trees. The distribution of a few key phylogenetic markers, such as contiguousness of cytochrome b, nature of the c-type subunit or spacing between b-heme ligands, are discussed. A localised modification of previous tree topologies is proposed on the basis of the obtained data. The comparison of extant enzymes furthermore allowed us to define the minimal functional and evolutionary core of the enzyme. The data furthermore suggest that the ancestral enzyme was put together from subunits that previously had played a role in other electron transfer chains.

Publisher Summary Since the beginning of 199Os, the trend appears to have reverted to strong interactions between the fields of photosynthetic studies and bioinorganic chemistry largely because of the impact of molecular biology, particularly, site-specific mutagenesis and heterologous expression. This chapter summarizes these recent advances with emphasis on the part played by iron–sulfur centers in photosynthesis. If the inorganic chemistry of iron–sulfur centers has evolved tremendously since 1962, the modern notion of photosynthesis equally has little to do with its early versions. It is known that plant-type photosynthesis occurred first in eubacteria or more specifically in the ancestors of extant cyanobacteria, and was subsequently imported into eukaryotes via the events of endosymbiosis. As no photosynthetic mechanisms based on chlorophylls have so far been found in archaebacteria, the evolution of photosynthesis is considered to have exclusively occurred within the domain of eubacteria. In this domain, two further (anoxygenic) photosynthetic principles have been observed, each involving a cytochrome bc complex and only a single reaction center (RC) that resembles either photosystem I or photosystem II of oxygenic photosynthesis. The iron–sulfur centers participating in the light reactions of the three different photosynthetic principles are discussed in the chapter.

N 4 -(2-Acetoxyethoxymethyl)-2-acetylpyridine thiosemicarbazone (AATSC) belongs to a series of molecules known to have broad antimicrobial inhibitory activity. These molecules contain the 2-acetoxyethoxy moiety which could conceivably take up a conformation analogous to that of the ribosyl group. Moreover, the thiosemicarbazone moiety, when it the presence of a suitable enzymatic site, could mimic the triazine group, which is found in a number of antifolate drugs. AATSC, which has both bacterial inhibitory activity and water solubility, was accordingly evaluated for its antifolate activity against the bovine liver dihydrofolate reductase. AATSC is shown to be a fully uncompetitive inhibitor of that enzyme. Furthermore, AATSC enhances the activity of methotrexate. Such a potentiation could be useful for therapeutic purposes.

The subunit structure and complete amino acid sequence of the lectin extracted from Lens culinaris (LcL) seeds was determined. In previous studies, the primary structure of the alpha-chain (Mr = 5,710) was shown to be homologous to the alpha-chain of the lectin from Pisum sativum, the Vicia cracca glucose-specific lectin, and a region in the middle of the concanavalin A sequence (residues 70-121). The complete amino acid sequence of the beta-chain (Mr = 17,572) has been determined from 11 tryptic peptides, 4 peptides derived by chemical cleavage of the beta-chain at its three tryptophan residues, 11 peptides obtained after digestion with Staphylococcus aureus protease, and 5 tryptic peptides from the succinylated polypeptide chain. The extensive homologies by alignment of the alpha- and beta-chains of the L. culinaris lectin with portions of concanavalin A situated between 1 to 45 and 70 to 237, suggest that the L. culinaris and Canavalia ensiformis lectins have evolved from each other. A comparison was made between the secondary structure of the C. ensiformis lectin and the probable secondary structure of the L. culinaris lectin as predicted by two different methods. The results indicate that the folding of these two polypeptides has been particularly well conserved during evolution. It is suggested that the L. culinaris lectin is synthesized as a single polypeptide chain and cleaved subsequently into two or possibly three fragments, two of which would be alpha and beta and the third a fragment homologous to portion 46 to 69 in concanavalin A. As in favin, the amino acids postulated to be involved in the formation of the hydrophobic cavity and the sugar and metal binding sites are highly conserved in L. culinaris lectin.

The structure of the heavy (H) form of the acetylcholine receptor, which comprises two covalently linked 250,000 Mr oligomers, has been investigated by numerical analysis of electron microscope images. Na-cholate solubilized Torpedo marmorata H-form receptor was reintegrated into artificial lipid vesicles and negatively stained with uranyl acetate prior to imaging in a conventional transmission microscope. The reconstituted preparations exhibited the standard polypeptide composition of the purified receptor (alpha 2 beta gamma delta) and the same transmembrane arrangement as in the native subsynaptic membrane. Covalent disulfide linkage between the two oligomers took place exclusively through the delta chains. In agreement with previous work (Cartaud et al., 1980) the H-form appeared as "doublets" of two coplanar 9 nm rosettes at a center-to-center distance of 9.2 +/- 1.1 nm. The relative angular orientation of the two rosettes in a doublet was examined by correlation analysis in the real space. It exhibited a marked variability, few of the doublets featuring any kind of symmetry, suggesting that the two oligomers of a doublets are connected via an extended and flexible chain or loop. The area of contact between the two rosettes of a doublet therefore does not necessarily represent a reliable clue as to the location of the delta chain within the structure. Averaged images obtained after reorientation and summation of up to 132 rosettes revealed the three major peaks and the two grooves already observed in previous studies. Two additional smaller peaks were identified. Tentative assignment of structural details to individual subunits was deduced from an examination of alpha-bungarotoxin-labeled doublets. The alpha subunits, which carry part or all of the acetylcholine binding sites, are probably located in nonadjacent positions in the vicinity of the newly found peaks. This assignment is consistent with the image analysis of receptor-toxin complexes recently reported by Zingsheim et al. (1982b).

The tertiary structure of Bacillus subtilis levansucrase (EC 2.4.1.10) at 3.8 A resolution has been obtained from x-ray diffraction data by the method of multiple isomorphous replacements using three heavy atom derivatives. The crystals belong to space group P212121 with 1 molecule/crystallographic asymmetric unit. Unit cell dimensions are: a = 53.8 A, b = 67.7 A, and c = 125.6 A. Isomorphous heavy atom derivatives were prepared by soaking crystals in solutions containing K2Hg(CN)4, KAu(CN)2, and K2PtCl6. Precession photographs were taken of 29 reciprocal lattice planes of the native compound and three derivatives. The intensities were measured with an automatic integrating microdensitometer. The heavy atoms were located by means of two-dimensional difference Patterson and Fourier projections. The heavy atom parameters were refined by a least squares method. In the subsequent phases of calculatin, a final mean figure of merit equal to 0.74 was obtained for the independent reflectins. A 3.8 A electron density map based on phases from three heavy atom derivatives has been calculated. The electron density map of levansucrase suggests that the molecule is a very elongated ellipsoid with overall dimensions of 26 X 32 X 117 A. A model of levansucrae is also presented.

Tat is a viral protein essential for activation of the HIV genes and plays an important role in the HIV-induced immunodeficiency. We chemically synthesized a Tat protein (86 residues) with its six glycines Ca labelled with C-13. This synthetic protein has the full Tat activity. Heteronuclear nuclear magnetic resonance (NMR) spectra and NOE back-calculation made;possible the sequential assignment of the 86 spin systems. Consequently, 915 NMR restraints were identified and 272 of them turned out to be long range ([i-j] > 4), providing structural information on the whole Tat protein. The poor spectral dispersion of Tat NMR spectra does not allow an accurate structure to be determined as for other proteins studied by 2D NMR. Nevertheless, we were able to determine the folding for Tat protein at a 1-mM protein concentration in a 100 mM, pH 4.5 phosphate buffer. The two main Tat functional regions, the basic region and the cysteine-rich region, are well exposed to solvent while a part of the N-terminal region and the C-terminal region constitute the core of Tat Bru. The basic region adopts an extended structure while the cysteine-rich region is made up of two loops. Resolution of this structure was determinant to develop a drug design approach against Tat. The chemical synthesis of the drugs allowed the specific binding and the inhibition of Tat to be verified. (C) 2000 Academie des sciences/Editions scientifiques et medicales Elsevier SAS. ispartof: C R Acad Sci III vol:323 issue:10 pages:883-894 ispartof: location:France status: published

The lentil lectin extracted from Lens culinaris seeds crystallizes in the orthorhombic space group P 2 1 2 1 2 1 with unit cell dimensions a =50·1 A, b =67·8 A and c =130·2 A. The asymmetric unit contains one, protein molecule. The crystals are suitable for high resolution work.