The origin,diversity, and structure function relationships of insect luciferases

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. Whereas the oxidative mechanism which leads to light emission is similar for most luciferases, these enzymes and their substrates are evolutionarily unrelated. Among all bioluminescent groups, insects constitute one of the most diverse in terms of biochemistry. In the fungus-gnats (Mycetophilidae: Diptera), for example, bioluminescence is generated by two biochemically distinct systems. Despite the diversity, investigations on insect luciferases and biochemistry have been conducted mostly with fireflies. The luciferases from the related phengodid beetles, which can produce green to red bioluminescence using the same chemistry as firefly luciferases, have been recently investigated. Beetle luciferases originated from ancestral acyl-CoA ligases. Present data suggest that conserved motifs among this class of ligases are involved in substrate adenylation. The three-dimensional structure of firefly luciferase was recently solved and mutagenesis studies have been performed identifying putative residues involved in luciferin binding and bioluminescence color determination in several beetle luciferases. The knowledge gained through these studies is helping in the development of useful reporter gene tools for biotechnological and biomedical purposes. Received 4 March 2002; received after revision 13 May 2002; accepted 21 May 2002     

The role of glycosylation in ionotropic glutamate receptor ligand binding, function, and trafficking

Members of the ionotropic glutamate receptor (iGluR) family have between 4 and 12 consensus asparagine (N)-linked glycosylation sites. They are localized on the extracellular N-termini, and the loop between the penultimate and last transmembrane domains. These regions also contain the essential elements for formation of the ligand binding site. N-linked glycosylation does not appear to be essential for formation of the ligand binding site per se, but there are demonstrated interactions between glycosylation state and ligand binding affinity, receptor physiology, susceptibility to allosteric modulation and, in some cases, trafficking. There is no indication of a general role for N-linked glycosylation in iGluRs; instead the effects of glycosylation vary among glutamate receptor subtypes and splice variants, with specific effects on structure or function with different subunits.     

Epimerases: structure, function and mechanism

Carbohydrates are ideally suited for molecular recognition. By varying the stereochemistry of the hydroxyl substituents, the simple six-carbon, six-oxygen pyranose ring can exist as 10 different molecules. With the further addition of simple chemical changes, the potential for generating distinct molecular recognition surfaces far exceeds that of amino acids. This ability to control and change the stereochemistry of the hydroxyl substituents is very important in biology. Epimerases can be found in animals, plants and microorganisms where they participate in important metabolic pathways such as the Leloir pathway, which involves the conversion of galactose to glucose-1-phosphate. Bacterial epimerases are involved in the production of complex carbohydrate polymers that are used in their cell walls and envelopes and are recognised as potential therapeutic targets for the treatment of bacterial infection. Several distinct strategies have evolved to invert or epimerise the hydroxyl substituents on carbohydrates. In this review we group epimerisation by mechanism and discuss in detail the molecular basis for each group. These groups include enzymes which epimerise by a transient keto intermediate, those that rely on a permanent keto group, those that eliminate then add a nucleotide, those that break then reform carbon-carbon bonds and those that linearize and cyclize the pyranose ring. This approach highlights the quite different biochemical processes that underlie what is seemingly a simple reaction. What this review shows is that each position on the carbohydrate can be epimerised and that epimerisation is found in all organisms.     

Arabinogalactan-proteins: structure, expression and function

Arabinogalactan-proteins (AGPs) are a family of extensively glycosylated hydroxyproline-rich glycoproteins that are thought to have important roles in various aspects of plant growth and development. After a brief introduction to AGPs highlighting the problems associated with defining and classifying this diverse family of glycoproteins, AGP structure is described in terms of the protein component (including data from molecular cloning), carbohydrate component, processing of AGPs (including recent data on glycosylphosphatidylinositol membrane anchors) and overall molecular shape. Next, the expression of AGPs is examined at several different levels, from the whole plant to the cellular levels, using a variety of experimental techniques and tools. Finally, AGP function is considered. Although the existing functional evidence is not incontrovertible, it does clearly point to roles for AGPs in vegetative, reproductive, and cellular growth and development as well as programmed cell death and social control. In addition and most likely inextricably linked to their functions, AGPs are presumably involved in molecular interactions and cellular signaling at the cell surface. Some likely scenarios are discussed in this context. AGPs also have functions of real or potential commercial value, most notably as emulsifiers in the food industry and as potential immunological regulators for human health. Several important questions remain to be answered with respect to AGPs. Clearly, elucidating the unequivocal functions of particular AGPs and relating these functions to their respective structures and modes of action remain as major challenges in the years ahead.     

Adducin: structure, function and regulation

Adducin is a ubiquitously expressed membrane-skeletal protein localized at spectrin-actin junctions that binds calmodulin and is an in vivo substrate for protein kinase C (PKC) and Rho-associated kinase. Adducin is a tetramer comprised of either alpha/beta or alpha/gamma heterodimers. Adducin subunits are related in sequence and all contain an N-terminal globular head domain, a neck domain and a C-terminal protease-sensitive tail domain. The tail domains of all adducin subunits end with a highly conserved 22-residue myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that has homology to MARCKS protein. Adducin caps the fast-growing ends of actin filaments and also preferentially recruits spectrin to the ends of filaments. Both the neck and the MARCKS-related domains are required for these activities. The neck domain self-associates to form oligomers. The MARCKS-related domain binds calmodulin and contains the major phosphorylation site for PKC. Calmodulin, gelsolin and phosphorylation by the kinase inhibit in vitro activities of adducin involving actin and spectrin. Recent observations suggest a role for adducin in cell motility, and as a target for regulation by Rho-dependent and Ca2+-dependent pathways. Prominent physiological sites of regulation of adducin include dendritic spines of hippocampal neurons, platelets and growth cones of axons.     

The structure and function of platelet-activating factor acetylhydrolases

Platelet-activating factor acetylhydrolases (PAF-AHs, EC 3.1.1.47) constitute a unique and biologically important family of phospholipase A₂s. They are related to neither the well-characterized secretory nor cytosolic PLA₂s, and unlike them do not require Ca²⁺ for catalytic activity. The distinguishing property of PAF-AHs is their unique substrate specificity they act on the phospholipid platelet-activating factor (PAF), and in some cases on proinflammatory polar phospholipids, from which they remove a short acyl moiety – acetyl in the case of PAF – located at the sn-2 position. Because PAF is found both in the plasma and in the cytosol of many tissues, PAF-acetylhydrolases are equally widely distributed in an animal organism. Recent crystallographic studies shed new light on the complex structure-function relationships in PAF-AHs. Received 15 September 1997; received after revision 23 February 1998; accepted 25 February 1998     

Homing endonucleases: structure, function and evolution

‘Homing’ is the lateral transfer of an intervening genetic sequence, either an intron or an intein, to a cognate allele that lacks that element. The end result of homing is the duplication of the intervening sequence. The process is initiated by site-specific endonucleases that are encoded by open reading frames within the mobile elements. Several features of these proteins make them attractive subjects for structural and functional studies. First, these endonucleases, while unique, may be contrasted with a variety of enzymes involved in nucleic acid strand breakage and rearrangement, particularly restriction endonucleases. Second, because they are encoded within the intervening sequence, there are interesting limitations on the position and length of their open reading frames, and therefore on their structures. Third, these enzymes display a unique strategy of flexible recognition of very long DNA target sites. This strategy allows these sequences to minimize nonspecific cleavage within the host genome, while maximizing the ability of the endonuclease to cleave closely related variants of the homing site. Recent studies explain a great deal about the biochemical and genetic mechanisms of homing, and also about the structure and function of several representative members of the homing endonuclease families. Received 6 January 1999; received after revision 24 February 1999; accepted 24 February 1999     

Inositol pyrophosphates: structure, enzymology and function

The stereochemistry of the inositol backbone provides a platform on which to generate a vast array of distinct molecular motifs that are used to convey information both in signal transduction and many other critical areas of cell biology. Diphosphoinositol phosphates, or inositol pyrophosphates, are the most recently characterized members of the inositide family. They represent a new frontier with both novel targets within the cell and novel modes of action. This includes the proposed pyrophosphorylation of a unique subset of proteins. We review recent insights into the structures of these molecules and the properties of the enzymes which regulate their concentration. These enzymes also act independently of their catalytic activity via protein–protein interactions. This unique combination of enzymes and products has an important role in diverse cellular processes including vesicle trafficking, endo- and exocytosis, apoptosis, telomere length regulation, chromatin hyperrecombination, the response to osmotic stress, and elements of nucleolar function.     

Serine peptidases: Classification, structure and function

Serine peptidases play key roles in human health and disease and their biochemical properties shaped the molecular evolution of these processes. Of known proteolytic enzymes, the serine peptidase family is the major cornerstone of the vertebrate degradome. We describe the known diversity of serine peptidases with respect to structure and function. Particular emphasis is placed on the S1 peptidase family, the trypsins, which underwent the most predominant genetic expansion yielding the enzymes responsible for vital processes in man such as digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. Received 13 December 2007; received after revision 8 January 2008; accepted 22 January 2008     

Convergent evolution of defensin sequence,structure and function

Defensins are a well-characterised group of small, disulphide-rich, cationic peptides that are produced by essentially all eukaryotes and are highly diverse in their sequences and structures. Most display broad range antimicrobial activity at low micromolar concentrations, whereas others have other diverse roles, including cell signalling (e.g. immune cell recruitment, self/non-self-recognition), ion channel perturbation, toxic functions, and enzyme inhibition. The defensins consist of two superfamilies, each derived from an independent evolutionary origin, which have subsequently undergone extensive divergent evolution in their sequence, structure and function. Referred to as the cis- and trans-defensin superfamilies, they are classified based on their secondary structure orientation, cysteine motifs and disulphide bond connectivities, tertiary structure similarities and precursor gene sequence. The utility of displaying loops on a stable, compact, disulphide-rich core has been exploited by evolution on multiple occasions. The defensin superfamilies represent a case where the ensuing convergent evolution of sequence, structure and function has been particularly extreme. Here, we discuss the extent, causes and significance of these convergent features, drawing examples from across the eukaryotes.     

The structure and function of initiation factors in eukaryotic protein synthesis

Protein synthesis is one of the most complex cellular processes, involving numerous translation components that interact in multiple sequential steps. The most complex stage in protein synthesis is the initiation process. It involves initiation factor-mediated assembly of a 40S ribosomal subunit and initiator tRNA into a 48S initiation complex at the initiation codon of an mRNA and subsequent joining of a 60S ribosomal subunit to form a translationally active 80S ribosome. The basal set of factors required for translation initiation has been determined, and biochemical, genetic, and structural studies are now beginning to reveal details of their individual functions in this process. The mechanism of translation initiation has also been found to be influenced significantly by structural properties of the 5' and 3' termini of individual mRNAs. This review describes some of the major developments in elucidating molecular details of the mechanism of initiation that have occurred over the last decade.     

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.     

Protein structure and function

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Protein structure and function