Tauropine dehydrogenase from the marine sponge Halichondria japonica is a homolog of ornithine cyclodeaminase/mu-crystallin

The partial amino acid sequence including the N- and C-terminal portions of tauropine dehydrogenase (EC 1.5.1.23) from the marine sponge Halichondria japonica was determined by enzymatic cleavages followed by peptide sequencing. This information was used to design degenerate primers for amplification of cDNA encoding the tauropine dehydrogenase. The cDNA included 1231 nucleotides with an open reading frame of 1002 nucleotides that encodes a protein of 334 amino acid residues. From the peptide and nucleotide sequencing, the mature tauropine dehydrogenase was estimated to consist of 333 amino acid residues with an acetylated N-terminal serine residue and no intrachain disulfide bonds. The primary structure of the H. japonica enzyme showed apparent similarity with a homolog of ornithine cyclodeaminase from Rhizobium meliloti and other proteins of the ornithine cyclodeaminase/mu-crystallin family, but it showed no significant similarity with the known sequences of octopine dehydrogenases and tauropine dehydrogenases from marine invertebrates. These findings indicate that opine dehydrogenases in marine invertebrates are not all homologous.     

Ornithine cyclodeaminase from Ti plasmid C58: DNA sequence, enzyme properties and regulation of activity by arginine

Nopaline, an abundant opine in plant cells transformed with nopaline-type Ti plasmids, is catabolized in Agrobacterium by three Ti-plasmid-coded steps via arginine and ornithine to proline. The last enzyme, ornithine cyclodeaminase (OCD), converts ornithine directly into proline with release of ammonia. We describe the DNA sequence of the ocd gene from Ti plasmid C58, antiserum against an OCD fusion protein overexpressed in Escherichia coli, induction and identification of the gene product in Agrobacterium and enzymatic properties of the protein. The DNA sequence suggests a soluble protein with a stretch of some homology with ornithine carbamoyltransferases from other bacteria. OCD activity is subject to substrate inhibition, is stimulated by NAD+ (presumably acting as a catalytic cofactor) and is regulated by L-arginine which has pronounced effects on the optima for pH and temperature and on the Km for ornithine. The regulation of OCD activity by L-arginine is discussed as part of the mechanisms which integrate the pathway of Ti-plasmid-coded opine utilization with general metabolism in Agrobacterium.     

Thermus thermophilus glycoside hydrolase family 57 branching enzyme: crystal structure, mechanism of action, and products formed

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)₇-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.     

Transketolase: structure and mechanism of action

The structure and mechanism of action of transketolase are reviewed, with the primary emphasis laid on the baker's yeast enzyme. The oligomeric structure of transketolase, the interaction of the coenzyme with the apoenzyme and the role of phosphate groups in the substrate interaction with the protein have been studied. The role of essential groups of apotransketolase in the binding of the coenzyme, substrates as well as in the catalytic act are described. The peculiarities of formation of the enzyme active center are discussed.     

Sequence similarity between cholera toxin and glycoprotein hormones: implications for structure activity relationship and mechanism of action.

The B chain of cholera toxin and the β subunits of thyrotropin, luteinizing hormone, human chorionic gonadotropin, and follicle-stimulating hormone are shown to have a region of sequence analogy believed to correlate with their ability to bind to receptors on cell membranes. A possible sequence analogy is also defined in the α subunits of these glycoprotein hormones and a region of the cholera toxin A₁ chain believed to be responsible for adenylate cyclase activation.     

Site-directed mutagenesis of ricin A-chain and implications for the mechanism of action

Ricin A-chain is an N-glycosidase that attacks ribosomal RNA at a highly conserved adenine residue. The enzyme is representative of a large family of medically significant proteins used in the design of anticancer agents and in the treatment of HIV infection. The x-ray structure has been used as a guide to create several active site mutations by directed mutagenesis of the cloned gene. Glu177 is a key catalytic residue, and conversion to Gln reduces activity 180-fold. Asn209 is shown to participate in substrate binding by kinetic analysis. Conversion to Ser increases Km sixfold but has no effect on kcat. Conversion of Tyr80 and Tyr123 to Phe decreases activity by 15- and 7-fold respectively. A mechanism of action is proposed that involves binding of the substrate adenine in a syn configuration that resembles the transition state; the putative oxycarbonium ion is probably stabilized by interaction with Glu177.     

Energetics of RNA cleavage: implications for the mechanism of action of ribozymes.

A new class of ribozymes produce 2',3'-cyclic phosphate upon self-catalyzed cleavage of RNA molecules, similar to those observed during enzymatic (RNase-catalyzed) as well as non-enzymatic hydrolyses of RNAs. This product suggests that the reaction intermediate/transition state is a pentacoordinated oxyphosphorane. In order to elucidate the energetics of these RNA cleaving reactions, the reaction coordinate has been simulated and a pentacoordinated intermediate has been characterized via ab initio molecular orbital calculations utilizing the dianionic hydrolysis-intermediate of methyl ethylene phosphate as a model compound. The calculated reaction coordinate indicates that the transition state for the P-O(2') bond cleavage is lower in energy than that for the P-O(5') bond cleavage under uncatalyzed conditions. Thus, the dianionic pentacoordinated phosphorus intermediate tends to revert back to the starting RNA by cleaving the P-O(2') bond rather than productively cleaving the P-O(5') bond. In order for ribozymes to effectively cleave RNA molecules, it is therefore mandatory to stabilize the leaving 5'-oxygen, e.g. by means of a divalent magnesium ion.     

The 2-hydroxycarboxylate transporter family: physiology, structure, and mechanism.

The 2-hydroxycarboxylate transporter family is a family of secondary transporters found exclusively in the bacterial kingdom. They function in the metabolism of the di- and tricarboxylates malate and citrate, mostly in fermentative pathways involving decarboxylation of malate or oxaloacetate. These pathways are found in the class Bacillales of the low-CG gram-positive bacteria and in the gamma subdivision of the Proteobacteria. The pathways have evolved into a remarkable diversity in terms of the combinations of enzymes and transporters that built the pathways and of energy conservation mechanisms. The transporter family includes H+ and Na+ symporters and precursor/product exchangers. The proteins consist of a bundle of 11 transmembrane helices formed from two homologous domains containing five transmembrane segments each, plus one additional segment at the N terminus. The two domains have opposite orientations in the membrane and contain a pore-loop or reentrant loop structure between the fourth and fifth transmembrane segments. The two pore-loops enter the membrane from opposite sides and are believed to be part of the translocation site. The binding site is located asymmetrically in the membrane, close to the interface of membrane and cytoplasm. The binding site in the translocation pore is believed to be alternatively exposed to the internal and external media. The proposed structure of the 2HCT transporters is different from any known structure of a membrane protein and represents a new structural class of secondary transporters.     

细菌sRNA ArcZ结构、功能及作用机制研究进展

ArcZ是一种大小为121个核苷酸的细菌非编码反式小RNA分子(small noncoding RNA,sRNA)。通过激活rpoS的表达,ArcZ间接地促进生物被膜基体组成部分菌毛和纤维素的表达;另外,其与ArcA/ArcB双组分系统互相负调控从而影响细菌用氧环境。ArcZ在近几年的研究中已被确定为细菌毒力调节的sRNA,能够对多种毒力决定因子发挥多重调节,包括细菌活力、淀粉酶产出、生物被膜形成及Ⅲ型分泌系统。本研究综述了ArcZ的结构、功能及作用机制方面的研究进展,并对其存在的生理意义进行了探讨。     

Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism

By elevating the pH to 9.5 in 3 M KCl, the concentration of the N intermediate in the bacteriorhodopsin photocycle has been enhanced, and time-resolved resonance Raman spectra of this intermediate have been obtained. Kinetic Raman measurements show that N appears with a half-time of 4 +/- 2 ms, which agrees satisfactorily with our measured decay time of the M412 intermediate (2 +/- 1 ms). This argues that M412 decays directly to N in the light-adapted photocycle. The configuration of the chromophore about the C13 = C14 bond was examined by regenerating the protein with [12,14-2H]retinal. The coupled C12-2H + C14-2H rock at 946 cm-1 demonstrates that the chromophore in N is 13-cis. The shift of the 1642-cm-1 Schiff base stretching mode to 1618 cm-1 in D2O indicates that the Schiff base linkage to the protein is protonated. The insensitivity of the 1168-cm-1 C14-C15 stretching mode to N-deuteriation establishes a C = N anti (trans) Schiff base configuration. The high frequency of the C14-C15 stretching mode as well as the frequency of the 966-cm-1 C14-2H-C15-2H rocking mode shows that the chromophore is 14-s-trans. Thus, N contains a 13-cis, 14-s-trans, 15-anti protonated retinal Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous immunoregulatory peptides (myelopeptides): structure, function, and mechanism of action

Previously unknown bioregulators from bone marrow, myelopeptides, were isolated, identified, and synthesized, and their biological properties and mechanism of action were studied in detail. Phe-Leu-Gly-Phe-Pro-Thr (MP-1) manifests an immunocorrecting effect by restoring the level of antibody production in animals suffering from immunodeficiencies of various etiologies; Leu-Val-Val-Tyr-Pro-Trp (MP-2) manifests an antitumor effect by abolishing the inhibitory action of tumor cells on the functional activity of T-lymphocytes; Leu-Val-Cys-Tyr-Pro-Gln (MP-3) stimulates phagocytosis by macrophages and, in this way, protects animals from infections caused by pathogenic microorganisms; and Phe-Pro-Arg-Ile-Met-Thr-Pro (MP-4) is a new factor of cell differentiation inducing terminal cell differentiation in the HL-60 and K-562 leukemic cell lines.     

Peptide methionine sulfoxide reductase: structure, mechanism of action, and biological function.

Reactive oxygen and nitrogen intermediates can cause damage to many cellular components and have been implicated in a number of diseases. Cells have developed a variety of mechanisms to destroy these reactive molecules or repair the damage once it occurs. In proteins one of the amino acids most easily oxidized is methionine, which is converted to methionine sulfoxide. An enzyme, peptide methionine sulfoxide reductase (MsrA), catalyzes the reduction of methionine sulfoxide in proteins back to methionine. There is growing evidence that MsrA plays an important role in protecting cells against oxidative damage. This paper reviews the biochemical properties and biological role of MsrA.     

Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action

Ribosomally synthesized peptides with antimicrobial activity are produced by prokaryotes, plants, and a wide variety of animals, both vertebrates and invertebrates. These peptides represent an important defense against micro-organisms. Although the peptides differ greatly in primary structures, they are nearly all cationic and very often amphiphilic, which reflects the fact that many of these peptides kill their target cells by permeabilizing the cell membrane. Moreover, many of these peptides may roughly be placed into one of three groups: (1) those that have a high content of one (or two) amino acid(s), often proline, (2) those that contain intramolecular disulfide bonds, often stabilizing a predominantly β-sheet structure, and (3) those with amphiphilic regions if they assume an α-helical structure. Most known ribosomally synthesized antimicrobial peptides have been identified and characterized during the past 15 years. As a result of these studies, insight has been gained into fundamental aspects of biology and biochemistry such as innate immunity, membrane-protein interactions, and protein modification and secretion. Moreover, it has become evident that these peptides may be developed into useful antimicrobial additives and drugs. This review presents a broad overview of the main types of ribosomally synthesized antimicrobial peptides produced by eukaryotes and prokaryotes. Received: 30 August 1996 / Accepted: 26 November 1996     

Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance

Host-defence peptides secreted from the skin glands of Australian frogs and toads, are, with a few notable exceptions, different from those produced by anurans elsewhere. This review summarizes the current knowledge of the following classes of peptide isolated and characterized from Australian anurans: neuropeptides (including smooth muscle active peptides, and peptides that inhibit the production of nitric oxide from neuronal nitric oxide synthase), antimicrobial and anticancer active peptides, antifungal peptides and antimalarial peptides. Other topics covered include sex pheromones of anurans, and the application of peptide profiling to (i). recognize particular populations of anurans of the same species and to differentiate between species, and (ii). investigate evolutionary aspects of peptide formation.     

Ornithine decarboxylase inhibitors increase the cellular content of the enzyme: implications for translational regulation

Ehrlich ascites tumor cells grown in the presence of inhibitors of ornithine decarboxylase (EC 4.1.1.17) exhibited an elevated content of this enzyme. The increase could not solely be explained by a decrease in the degradation rate of the enzyme. Instead a stimulation of enzyme synthesis, probably mediated via the polyamine-depleting properties of the inhibitors, is suggested. The enhancement of cellular ornithine decarboxylase content was not accompanied by any significant changes in the amount of ornithine decarboxylase mRNA, indicating a regulation at the level of translation.     

The structure of verdohemochrome and its implications for the mechanism of heme catabolism

The synthesis, purification as a tetrafluoroborate salt and structural elucidation of the verdohemochrome 2a derived from the coupled oxidation of octaethylhemochrome 1 is described. Based on elemental analyses, spectroscopic studies (visible and infrared absorption, ¹H-NMR) and fast atom bombardment mass spectrometry, the assignment of the iron(II) oxaporphyrin structure for the verdohemochrome 2a and the blue monocarbonyl species 2b, obtained upon treatment of 2a with carbon monoxide, has been accomplished. This assignment raises a number of questions regarding the iron oxidation state of intermediates in the pathway of heme catabolism both in vitro and in vivo. Furthermore, the implications of the occurrence of an iron oxaporphyrin intermediate in the pathway of heme metabolism, which is suggested by the similarity of the visible absorption spectrum of the CO species 2b with that of a new intermediate recently observed in the heme oxygenase-catalyzed degradition of heme and mesoheme, is considered.