Scapharca dimeric hemoglobin: A new mechanism of information transfer between globin chains

The homodimeric hemoglobin component present in the red cells of the bivalve molluscScapharca inaequivalvis, HbI, is endowed with high cooperativity in ligand binding. This behaviour is in contrast with that of vertebrate hemoglobins in which cooperativity is associated with a tetrameric assembly and the presence of two types of chain. Analysis of the aminoacid sequence and immunological data suggested that the assembly of HbI differed from that characteristic of vertebrate hemoglobins and hence that cooperativity had an unusual structural basis. Indeed the X-ray structures of the carbonmonoxy and deoxy derivatives at 2.4 resolution showed that in HbI the heme carrying E and F helices are not exposed to solvent as in the vertebrate hemoglobin tetramer, but form the subunit interface and bring the two heme groups practically in direct contact through a network of hydrogen bonds. Ligand binding brings about marked structural changes that are limited to the heme environment, whereas quaternary changes are only minor. The structural changes in the heme environment result in alterations in the network of interactions between the heme groups which lead to changes in ligand affinity. In HbI therefore cooperativity in ligand binding is achieved through direct heme-heme communication as opposed to the long range information transfer operative in the vertebrate hemoglobin tetramer.     

Biochemical characterization and bacterial expression of an odorant-binding protein from <Emphasis Type="Italic">Locusta migratoria</Emphasis>

Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100 degrees C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera.     

Sulfatase activities towards the regulation of cell metabolism and signaling in mammals

In higher vertebrates, sulfatases belong to a conserved family of enzymes that are involved in the regulation of cell metabolism and in developmental cell signaling. They cleave the sulfate from sulfate esters contained in hormones, proteins, and complex macromolecules. A highly conserved cysteine in their active site is post-translationally converted into formylglycine by the formylglycine-generating enzyme encoded by SUMF1 (sulfatase modifying factor 1). This post-translational modification activates all sulfatases. Sulfatases are extensively glycosylated proteins and some of them follow trafficking pathways through cells, being secreted and taken up by distant cells. Many proteoglycans, glycoproteins, and glycolipids contain sulfated carbohydrates, which are sulfatase substrates. Indeed, sulfatases operate as decoding factors for a large amount of biological information contained in the structures of the sulfated sugar chains that are covalently linked to proteins and lipids. Modifications to these sulfate groups have pivotal roles in modulating specific signaling pathways and cell metabolism in mammals.     

Cyclophosphamide-impaired regulation of hepatic heme metabolism

In male rats hepatic cytochromes b5 and P-450 were reduced at different times after treatment with cyclophosphamide (CP) (200 mg/kg i.p. for 3 days). In contrast, microsomal heme did not change until 48 h after the last dose of CP, leading to accumulation of heme in a 'non-cytochromal' form. Parallel to the above changes the heme metabolism showed derangement: delta-aminolaevulinate synthase, the rate-limiting enzyme in heme synthesis, was depressed and heme oxygenase, the enzyme which catalyzes the oxidative degradation of heme, was increased.     

Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters

Ferrochelatase (E.C. 4.99.1.1, protoheme ferrolyase) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme (heme). In the past 2 years, the crystal structures of ferrochelatases from the bacterium Bacillus subtilis and human have been determined. These structures along with years of biophysical and kinetic studies have led to a better understanding of the catalytic mechanism of ferrochelatase. At present, the complete DNA sequences of 45 ferrochelatases from procaryotes and eucaryotes are available. These sequences along with direct protein studies reveal that ferrochelatases, while related, vary significantly in amino acid sequence, molecular size, subunit composition, solubility, and the presence or absence of nitric-oxide-sensitive [2Fe-2S] cluster.     

S-Nitrosylation of proteins

The transfer of a nitric oxide group to cysteine sulfhydryls on proteins, known as S-nitrosylation, is increasingly becoming recognized as a ubiquitous regulatory reaction comparable to phosphorylation. It represents a form of redox modulation in diverse tissues, including the brain. An increasing number of proteins have been found to undergo S-nitrosylation in vivo. These proteins are called S-nitrosothiols, and may play an important role in many processes ranging from signal transduction, DNA repair, host defense, and blood pressure control to ion channel regulation and neurotransmission. This review focuses on the importance of the S-nitrosylation reaction and describes some recently identified S-nitrosothiols in various fields of research.     

[FeFe] hydrogenases and their evolution: a genomic perspective

Most hydrogenases (H2ases), the enzymes that produce or oxidize dihydrogen, possess dimetallic active sites and belong to either one of two phylogenetically distinct classes, the [NiFe] and the [FeFe] H2ases. These families of H2ases share a number of similarities regarding active site structure and reaction mechanism, as a result of convergent evolution. They are otherwise alien to each other, in particular with respect to protein sequence and structure, maturation mechanisms, and distribution among the realms of life. One of the interesting features of [FeFe] H2ases is their occurrence in anaerobic bacteria, anaerobic protists, and mitochondriate eukaryotes. They thus have the potential to report on important evolutionary events, including transitions from the prokaryote to the eukaryote lifestyle. Genome sequences yield a variety of [FeFe] H2ase sequences that have been implemented to shed light on the evolution of these proteins and their host organisms.     

The metabotropic GABA receptor: molecular insights and their functional consequences

Recent years have seen rapid and significant advances in our understanding of the G-protein-coupled gamma-amino butyric acid, B-type (GABA(B)) receptor, which could be a therapeutic target in conditions as diverse as epilepsy and hypertension. This progress originated with the ground-breaking work of Bernhard Bettler's team at Novartis who cloned the DNA encoding a GABA(B) receptor in 1997. Currently, the receptor is thought to be an unusual, possibly unique, example of a heterodimer composed of homologous, seven-transmembrane-domain (7TMD) subunits (named GABA(B) R1 and GABA(B) R2), neither of which is fully functional when expressed alone. The large N-terminal domain of the GABA(B) R1 subunit projects extracellularly and contains a ligand binding site. The similarity of the amino acid sequence of this region to some bacterial periplasmic amino acid-binding proteins of known structure has enabled structural and functional modelling of the N-terminal domain, and the identification of residues whose substitution modulates agonist/antagonist binding affinities. The intracellular C-terminal domains of the R1 and R2 subunits appear to constitute an important means of contact between the two subunits. Alternative splice variants, a common and functionally important feature of 7TMD proteins, have been demonstrated for the R1 subunit. Notably GABA(B) R1a differs from GABA(B) R1b by the possession of an N-terminal extension containing two complement protein modules (also called SCRs, or sushi domains) of unknown function. The levels at which each of the respective variants is expressed are not equal to one another, with variations occurring over the course of development and throughout the central nervous system. It is not yet clear, however, whether one variant is predominantly presynaptically located and the other postsynaptically located. The existence of as yet unidentified splice variants, additional receptor subtypes and alternative quaternary composition has not been ruled out as a source of receptor heterogeneity.     

Triosephosphate isomerase: a highly evolved biocatalyst

Triosephosphate isomerase (TIM) is a perfectly evolved enzyme which very fast interconverts dihydroxyacetone phosphate and d-glyceraldehyde-3-phosphate. Its catalytic site is at the dimer interface, but the four catalytic residues, Asn11, Lys13, His95 and Glu167, are from the same subunit. Glu167 is the catalytic base. An important feature of the TIM active site is the concerted closure of loop-6 and loop-7 on ligand binding, shielding the catalytic site from bulk solvent. The buried active site stabilises the enediolate intermediate. The catalytic residue Glu167 is at the beginning of loop-6. On closure of loop-6, the Glu167 carboxylate moiety moves approximately 2 Å to the substrate. The dynamic properties of the Glu167 side chain in the enzyme substrate complex are a key feature of the proton shuttling mechanism. Two proton shuttling mechanisms, the classical and the criss-cross mechanism, are responsible for the interconversion of the substrates of this enolising enzyme.     

Structure and function of type I copper in multicopper oxidases

The type I copper center in multicopper oxidases is constructed from 1Cys2His and weakly coordinating 1Met or the non-coordinating 1Phe/1Leu, and it exhibits spectral properties and an alkaline transition similar to those of the blue copper center in blue copper proteins. Since the type I copper center in multicopper oxidases is deeply buried inside the protein molecule, electron transfers to and from type I copper are performed through specific pathways: the hydrogen bond between an amino acid located at the substrate binding site and a His residue coordinating type I copper, and the His-Cys-His sequence connecting the type I copper center and the trinuclear copper center comprised of a type II copper and a pair of type III coppers. The intramolecular electron transfer rates can be tuned by mutating the fourth ligand of type I copper. Further, mutation at the Cys ligand gives a vacant type I copper center and traps the reaction intermediate during the four-electron reduction of dioxygen.     

Redox modulation of the NMDA receptor

Redox modulation has been recognized to be an important mechanism of regulation for the N-methyl-D-aspartate (NMDA) receptor. Sulfhydryl reducing agents enhance, whereas oxidizing agents decrease, NMDA-evoked currents. Multiple cysteine residues located in different NMDA receptor subunits have been identified as molecular determinants underlying redox modulation. The NMDA receptor is also regulated by nitric oxide (NO)-related species directly, not involving cyclic GMP, but the molecular mechanism of this action has heretofore not been entirely clear. The confusion arose at least partly due to the fact that various redox forms of NO (NO+, NO*, NO-, each having an additional electron compared with the previous) have distinct mechanisms of action. Recently, a critical cysteine residue (Cys 399) on the NR2A subunit has been shown to react under physiological conditions with NO by S-nitrosylation (transfer of the NO+ to cysteine thiol) or by reaction with NO- (nitroxyl anion) to underlie this form of modulation.     

Human sulfatases: A structural perspective to catalysis

The sulfatase family of enzymes catalyzes hydrolysis of sulfate ester bonds of a wide variety of substrates. Seventeen genes have been identified in this class of sulfatases, many of which are associated with genetic disorders leading to reduction or loss of function of the corresponding enzymes. Amino acid sequence homology suggests that the enzymes have similar overall folds, mechanisms of action, and bivalent metal ion-binding sites. A catalytic cysteine residue, strictly conserved in prokaryotic and eukaryotic sulfatases, is post-translationally modified into a formylglycine. Hydroxylation of the formylglycine residue by a water molecule forming the activated hydroxylformylglycine (a formylglycine hydrate or a gem-diol) is a necessary step for the enzyme's sulfatase activity. Crystal structures of three human sulfatases, arylsulfatases A and B(ARSA and ARSB), and estrone/dehydroepiandrosterone sulfatase or steroid sulfatase (STS), also known as arylsulfatase C, have been determined. While ARSA and ARSB are water-soluble enzymes, STS has a hydrophobic domain and is an integral membrane protein of the endoplasmic reticulum. In this article, we compare and contrast sulfatase structures and revisit the proposed catalytic mechanism in light of available structural and functional data. Examination of the STS active site reveals substrate-specific interactions previously identified as the estrogen-recognition motif. Because of the proximity of the catalytic cleft of STS to the membrane surface, the lipid bilayer has a critical role in the constitution of the active site, unlike other sulfatases.     

The presynaptic cytomatrix of brain synapses

Synapses are principal sites for communication between neurons via chemical messengers called neurotransmitters. Neurotransmitters are released from presynaptic nerve terminals at the active zone, a restricted area of the cell membrane situated exactly opposite to the postsynaptic neurotransmitter reception apparatus. At the active zone neurotransmitter-containing synaptic vesicles (SVs) dock, fuse, release their content and are recycled in a strictly regulated manner. The cytoskeletal matrix at the active zone (CAZ) is thought to play an essential role in the organization of this SV cycle. Several multi-domain cytoskeleton-associated proteins, including RIM, Bassoon, Piccolo/Aczonin and Munc-13, have been identified, which are specifically localized at the active zone and thus are putative molecular components of the CAZ. This review will summarize our present knowledge about the structure and function of these CAZ-specific proteins. Moreover, we will review our present view of how the exocytotic and endocytic machineries at the site of neurotransmitter release are linked to and organized by the presynaptic cytoskeleton. Finally, we will summarize recent progress that has been made in understanding how active zones are assembled during nervous system development.     

Plant glutaredoxins: still mysterious reducing systems

Glutaredoxins are ubiquitous oxidoreductases which are similar to thioredoxins and possess a typical glutathione-reducible CxxC or CxxS active site. We present here the current knowledge about these proteins in plants. At least 31 glutaredoxin genes are present in Arabidopsis thaliana, a value close to the thioredoxin gene number. Based essentially on active site sequences, a classification of these multiple genes is proposed. The specificity of the various apparently redundant forms within the glutaredoxin group or between glutaredoxin and thioredoxin can be analysed in terms of differential spatiotemporal expression of the genes, specificity vs. target proteins and mode of catalysis (glutathiolation/ deglutathiolation processes appear to be a specific function of glutaredoxin). Additional putative functions are proposed for plant glutaredoxins based on their targets in other organisms and in the light of the existence of hybrid proteins containing glutaredoxin modules in their N- or C-terminal part.Received 31 October 2003; received after revision 26 November 2003; accepted 4 December 2003     

The role of the cysteinyl and one of the tryptophyl residues in the neurotoxic action of suberitine

Summary The selective photooxidation of the single cysteinyl residue and one of the 3 tryptophyl residues of suberitine has been performed by irradiation using crystal violet and proflavine respectively as photosensitizers. Crystal violet and the protein form a 11 complex with a consequent partial inhibition of the neurotoxic activity. The latter is completely abolished by specific photooxidation of cysteine which is probably involved in the active site of the protein. The modification of the tryptophyl residue induces a large loss of the activity as a consequence of a photoinduced extensive denaturation of suberitine.     

L'azione protettiva dell'acido tioctico nell'intossicazione da cianuro di potassio

Summary The protective action of thioctic acid and of its reduced form in acute potassium cyanide poisoning has been studied in comparison with the antitoxic action of other compounds with mercapto or disulfide groups.The results permit the following conclusions: (1) Maximum protective action is given by reduced thioctic acid; (2) thioctic acid and BAL have almost the same activity; (3) cysteine and sodium thiosulfate have a protective activity of a much lower order; (4) panthethine is almost inactive. The authors offer the hypothesis that the greater activity of thioctic acid is due to its possible presence as coenzyme in rhodanese.     

Transthyretin: a review from a structural perspective

Transthyretin (formerly called prealbumin) plays important physiological roles as a transporter of thyroxine and retinol-binding protein. X-ray structural studies have provided information on the active conformation of the protein and the site of binding of both ligands. Transthyretin is also one of the precursor proteins commonly found in amyloid deposits. Both wild-type and single-amino-acid-substituted variants have been identified in amyloid deposits, the variants being more amyloidogenic. Sequencing of the gene and the resulting production of a transgenic mouse model have resulted in progress toward solving the mechanism of amyloid formation and detecting the variant gene in individuals at risk. Received 23 January 2001; received after revision 4 April 2001; accepted 30 April 2001     

Heme structure in cooperative and noncooperative hemoglobins: study by resonant Raman diffusion]

The modifications of heme sites of hemoglobin, which should occur upon apoprotein alterations (responsible for variations of oxygen affinity), have been examined by Resonnant Raman scattering. The oxygenated (R) and deoxygenated (T) shape of apoprotein do not modify the heme states. The spectral differences between these forms are essentially due to the presence or the absence of the sixth ligand.     

Canonical protein inhibitors of serine proteases

Serine proteases and their natural protein inhibitors are among the most intensively studied protein complexes. About 20 structurally diverse inhibitor families have been identified, comprising -helical, sheet, and / proteins, and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished based on their mechanism of action: canonical (standard mechanism) and non-canonical inhibitors, and serpins. The canonical inhibitors bind to the enzyme through an exposed convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is always very similar and resembles that of an ideal substrate. The non-canonical inhibitors interact through their N-terminal segment. There are also extensive secondary interactions outside the active site, contributing significantly to the strength, speed, and specificity of recognition. Serpins, similarly to the canonical inhibitors, interact with their target proteases in a substrate-like manner; however, cleavage of a single peptide bond in the binding loop leads to dramatic structural changes.Received 28 March 2003; received after revision 12 May 2003; accepted 16 May 2003     

Type-2 copper-containing enzymes

Type-2 Cu sites are found in all the major branches of life and are often involved in the catalysis of oxygen species. Four type-2 Cu protein families are selected as model systems for review: amine oxidases, Cu monooxygenases, nitrite reductase/multicopper oxidase, and CuZn superoxide dismutase. For each model protein, the availability of multiple crystal structures and detailed enzymological studies provides a detailed molecular view of the type-2 Cu site and delineation of the mechanistic role of the Cu in biological function. Comparison of these model proteins leads to the identification of common properties of the Cu sites and insight into the evolution of the trinuclear active site found in multicopper oxidases. Received 6 July 2007; accepted 18 July 2007