Structure and function of eukaryotic NAD(P)H:nitrate reductase

Pyridine nucleotide-dependent nitrate reductases (NRs; EC 1.6.6.1–3) are molybdenum-containing enzymes found in eukaryotic organisms which assimilate nitrate. NR is a homodimer with an ∼100 kDa polypeptide which folds into stable domains housing each of the enzyme's redox cofactors—FAD, heme-Fe molybdopterin (Mo-MPT) and the electron donor NAD(P)H—and there is also a domain for the dimer interface. NR has two active sites: the nitrate-reducing Mo-containing active site and the pyridine nucleotide active site formed between the FAD and NAD(P)H domains. The major barriers to defining the mechanism of catalysis for NR are obtaining the detailed three-dimensional structures for oxidized and reduced enzyme and more in-depth analysis of electron transfer rates in holo-NR. Recombinant expression of holo-NR and its fragments, including site-directed mutagenesis of key acative site and domain interface residues, are expected to make large contributions to this effort to understand the catalytic mechanism of NR.     

Mechanism-based targeting of NMDA receptor functions

NMDA receptors (NRs) are key signaling proteins in the central nervous system and represent important targets for drug development in several neurologic disorders. They are critically involved with fundamental brain processes, and thus indiscriminate pharmacological suppression of NR currents has seen only modest therapeutic success so far. Targeting harmful NR receptor activities while sparing the receptor’s vital functions requires a better understanding of the complexity of NR activation reaction; of the range of mechanisms that modulate discrete receptor activities; and of the consequences of this modulation on specific receptor functions. A quantitative account of the NR activation pathway was recently proposed and validated. It describes the gating reaction as a sequential, multi-step process rather than a binary, on-off switch. Alongside isoform-specific modulators, state-specific modulators may represent sophisticated interventions with high potential for narrow, functional specifi city. Here I review physiologic mechanisms that control NR responses; the salient features of the NR activation reaction; and discuss the model’s validity and its implications for drug development and characterization.Submitted 25 May 2005; accepted 29 June 2005     

Introduction: the ribonuclease A superfamily

In this multi-author issue several aspects of the ribonuclease A superfamily are reviewed. This superfamily can be subdivided in a number of mammalian and other vertebrate ribonuclease families. In the introduction chapter the titles of the other contributions are presented. There is little uniformity in the nomenclature of ribonucleases, caused in part by gene duplications, which have occurred independently in several mammalian lineages, and which are nice examples for explaining orthology and paralogy in molecular evolution.     

Turning points in the evolution of peroxidase–catalase superfamily: molecular phylogeny of hybrid heme peroxidases

Heme peroxidases and catalases are key enzymes of hydrogen peroxide metabolism and signaling. Here, the reconstruction of the molecular evolution of the peroxidase–catalase superfamily (annotated in pfam as PF00141) based on experimentally verified as well as numerous newly available genomic sequences is presented. The robust phylogenetic tree of this large enzyme superfamily was obtained from 490 full-length protein sequences. Besides already well-known families of heme b peroxidases arranged in three main structural classes, completely new (hybrid type) peroxidase families are described being located at the border of these classes as well as forming (so far missing) links between them. Hybrid-type A peroxidases represent a minor eukaryotic subfamily from Excavates, Stramenopiles and Rhizaria sharing enzymatic and structural features of ascorbate and cytochrome c peroxidases. Hybrid-type B peroxidases are shown to be spread exclusively among various fungi and evolved in parallel with peroxidases in land plants. In some ascomycetous hybrid-type B peroxidases, the peroxidase domain is fused to a carbohydrate binding (WSC) domain. Both here described hybrid-type peroxidase families represent important turning points in the complex evolution of the whole peroxidase–catalase superfamily. We present and discuss their phylogeny, sequence signatures and putative biological function.     

Tetraspanins

The first tetraspanins were discovered on surface of human leucocytes, but it was rapidly demonstrated that they had a wider tissue expression. Twenty-six molecules display sufficient homology to belong to the same superfamily. Their function is not precisely known, but data coming from biochemical studies or knockout mice suggest that they play a major role in membrane biology. One of their outstanding properties is their ability to form a network of multimolecular complexes, the 'tetraspanin web', in which integrins are included. The structure of these complexes is under investigation, but some of the rules that govern their organization have already been unraveled. The challenge is to determine how the organization of the 'tetraspanin web' modifies the function of its constitutive molecules and consequently influences cellular behaviour. The implications may be considerable for the understanding of basic cellular processes such as migration and also of diseases related to loss or mutation of a single tetraspanin. Received 29 December 2000; received after revision 26 February 2001; accepted 19 March 2001     

On the existence of cytokines in invertebrates

Based on the assumption that invertebrates, like vertebrates, possess factors regulating responses to infection or wounding, studies dealing with the evolution of immunity have focussed on the isolation and characterisation of putative cytokine-related molecules from invertebrates. Until recently, most of our knowledge of cytokine- and cytokine receptor-like molecules in invertebrates relies on functional assays and similarities at the physicochemical level. As such, a phylogenetic relationship between invertebrate cytokine-like molecules and vertebrate counterparts could not be convincingly demonstrated. Recent genomic sequence analyses of interleukin-1-receptor-related molecules, that is Toll-like receptors, and members of the transforming growth factor-β superfamily suggest that the innate immune system of invertebrates and vertebrates evolved independently. In addition, data from protochordates and annelids suggest that invertebrate cytokine-like molecules and vertebrate factors do not have the same evolutionary origin. We propose instead that the convergence of function of invertebrate cytokine-like molecules and vertebrate counterparts involved in innate immune defences may be based on similar lectin-like activities. Received 27 November 2000; received after revision 11 December 2000; accepted 13 December 2000     

Structure and evolution of the genetic code viewed from the perspective of the experimentally expanded amino acid repertoire in vivo

Much effort has been devoted recently to expanding the amino acid repertoire in protein biosynthesis in vivo. From such experimental work it has emerged that some of the non-canonical amino acids are accepted by the cellular translational machinery while others are not, i.e. we have learned that some determinants must exist and that they can even be anticipated. Here, we propose a conceptual framework by which it should be possible to assess deeper levels of the structure of the genetic code, and based on this experiment to understand its evolution and establishment. First, we propose a standardised repertoire of 20 amino acids as a basic set of conserved building blocks in protein biosynthesis in living cells to be the main criteria for genetic code structure and evolutionary considerations. Second, based on such argumentation, we postulate the structure and evolution of the genetic code in the form of three general statements: (i) the nature of the genetic code is deterministic; (ii) the genetic code is conserved and universal; (iii) the genetic code is the oldest known level of complexity in the evolution of living organisms that is accessible to our direct observation and experimental manipulations. Such statements are discussed as our working hypotheses that are experimentally tested by recent findings in the field of expanded amino acid repertoire in vivo. Received 30 June 1999; accepted 9 July 1999     

Eye lens development and gamma crystallins in Discoglossus pictus (Anura).

The ontogeny and localization of the gamma crystallins in Discoglossus pictus lens development has been determined. Using antibody specific for amphibian gamma crystallins in the immunofluorescence technique, it was found that gamma crystallins first appear in primary lens fibre cells in the lens rudiment, and continue to be restricted to the fibre area as lens development progresses. Thus the role of gamma crystallins as indicators of a differentiated state remains constant in amphibian evolution, having been demonstrated in the most archaic anuran superfamily, as well as in others more recently evolved.     

Common structural traits for cystine knot domain of the TGFβ superfamily of proteins and three-fingered ectodomain of their cellular receptors

The transforming growth factor-β (TGFβ) superfamily of proteins and their receptors are crucial developmental factors for all metazoan organisms. Cystine-knot (CK) motif is a spatial feature of the TGFβ superfamily of proteins whereas the extra-cellular domains (ectodomains) of their respective receptors form three-fingered protein domain (TFPD), both stabilized by tight cystine networks. Analyses of multiple sequence alignments of these two domains encoded in various genomes revealed that the cystines forming the CK and TFPD folds are conserved, whereas the remaining polypeptide patches are diversified. Orthologues of the human TGFβs and their respective receptors expressed in diverse vertebrates retain high sequence conservation. Examination of 3D structures of various TGFβ factors bound to their receptors have revealed that the CK and TFPD domains display several similar spatial traits suggesting that these two different protein folds might have been acquired from a common ancestor.     

Plant mitochondrial carriers: an overview

In the two last decades, biochemical studies using mitochondrial swelling experiments or direct solute uptake in isolated mitochondria have lead to the identification of different transport systems at the level of the plant mitochondrial inner membrane. Although most of them have been found to have similar features to those identified in animal mitochondria, some differences have been observed between plant and animal transporters. More recently, molecular biology studies have revealed that most of the mitochondrial exchanges are performed by nuclear encoded proteins, which form a superfamily. Members of this family have been reported in animals, yeast as well as plants. This review attempts to give an overview of the present knowledge concerning the biochemical and molecular characterisation of plant members of the mitochondrial carrier family and, when possible, a comparison with carriers from other organisms.     

Structure to function relationships in ceruloplasmin: a 'moonlighting' protein

Specialised copper sites have been recruited during evolution to provide long-range electron transfer reactivity and oxygen binding and activation in proteins destined to cope with oxygen reactivity in different organisms. Ceruloplasmin is an ancient multicopper oxidase evolved to insure a safe handling of oxygen in some metabolic pathways of vertebrates. The presently available knowledge of its structure provides a glimpse of its plasticity, revealing a multitude of binding sites that point to an elaborate mechanism of multifunctional activity. Ceruloplasmin represents an example of a 'moonlighting' protein that overcomes the one gene-one structure-one function concept to follow the changes of the organism in its physiological and pathological conditions. Received 19 February 2002; received after revision 29 March 2002; accepted 2 April 2002 RID="*" ID="*"Corresponding author.