Cloning, expression, purification, and characterization of Leishmania major dihydroorotate dehydrogenase

Leishmania major Friedlin (LmjF) is a protozoan parasite whose genomic sequence has been recently elucidated. Here we have cloned, overexpressed, purified, and characterized the product of the gene from LmjF chromosome 16: LmjF16.0530, which encodes a protein with putative dihydroorotate dehydrogenase activity. Dihydroorotate dehydrogenase (DHODH) is a flavoprotein that catalyses the oxidation of L-dihydroorotate to orotate, the fourth sequential step in the de novo pyrimidine nucleotide synthesis pathway. The predicted enzyme from L. major was cloned and expressed in Escherichia coli strain BL21(DE3) as a histidine-tag fusion protein and purified to homogeneity using affinity chromatography. The final product was homogeneous in SDS-PAGE gel electrophoresis. The dihydroorotate oxidase activity has been assayed and the steady-state kinetic mechanism has been determined using fumarate as the oxidizing substrate. The catalysis by LmDHODH enzyme proceeds by a Ping-Pong Bi-Bi mechanism and the kinetic parameters Km were calculated to be 90 and 418 microM for dihydroorotate and fumarate, respectively, and Vmax was calculated to be 11 micromol min-1 mg-1. Our results confirmed that the product of the gene LmjF16.0530, whose function has previously been predicted based on homology to known proteins, can therefore be positively assigned as L. major DHODH.     

Energy, Life, and ATP

The mechanism by which ATP is synthesized during oxidative and photophosphorylation has been elucidated by oxygen exchange and other studies: a novel form of catalysis--termed rotary catalysis--is involved.     

3D domain swapping, protein oligomerization, and amyloid formation

In 3D domain swapping, first described by Eisenberg, a structural element of a monomeric protein is replaced by the same element from another subunit. This process requires partial unfolding of the closed monomers that is then followed by adhesion and reconstruction of the original fold but from elements contributed by different subunits. If the interactions are reciprocal, a closed-ended dimer will be formed, but the same phenomenon has been suggested as a mechanism for the formation of open-ended polymers as well, such as those believed to exist in amyloid fibrils. There has been a rapid progress in the study of 3D domain swapping. Oligomers higher than dimers have been found, the monomer-dimer equilibrium could be controlled by mutations in the hinge element of the chain, a single protein has been shown to form more than one domain-swapped structure, and recently, the possibility of simultaneous exchange of two structural domains by a single molecule has been demonstrated. This last discovery has an important bearing on the possibility that 3D domain swapping might be indeed an amyloidogenic mechanism. Along the same lines is the discovery that a protein of proven amyloidogenic properties, human cystatin C, is capable of 3D domain swapping that leads to oligomerization. The structure of domain-swapped human cystatin C dimers explains why a naturally occurring mutant of this protein has a much higher propensity for aggregation, and also suggests how this same mechanism of 3D domain swapping could lead to an open-ended polymer that would be consistent with the cross-beta structure, which is believed to be at the heart of the molecular architecture of amyloid fibrils.     

Structure,function, and mechanism of HhaI DNA methyltransferases

A vast amount of literature has accumulated on the characterization of DNA methyltransferases. The HhaI DNA methyltransferase, a C5-cytosine methyltransferase, has been the subject of investigation for the last 2 decades. Biochemical and kinetic characterization have led to an understanding of the catalytic and kinetic mechanism of the methyltransfer reaction. The HhaI methyltransferase has also been subjected to extensive structural analysis, with the availability of 12 structures with or without a cofactor and a variety of DNA substrates. The mechanism of base flipping, first described for the HhaI methyltransferase, is conserved among all DNA methyltransferases and is also found to occur in numerous DNA repair enzymes. Studies with other methyltransferase reveal a significant structural and functional similarity among different types of methyltransferases. This review aims to summarize the available information on the HhaI DNA methyltransferase.     

Relationship between interferon-gamma, indoleamine 2,3-dioxygenase, and tryptophan catabolism

Interferons have been shown to be potential anti-cancer agents and to inhibit tumor cell growth in culture. The in vivo mechanism of the anti-proliferative effect may be direct or indirect through the immune system; however, in vitro a primary mechanism of cytotoxicity is through the depletion of tryptophan. In particular, interferon-gamma (IFN-gamma) induces an enzyme of tryptophan catabolism, indoleamine 2,3-dioxygenase (IDO), which is responsible for conversion of tryptophan and other indole derivatives to kynurenine. The inhibitory effect of interferon on many intracellular parasites such as Toxoplasma gondii and Chlamydia trachomatis is by the same mechanism. Elevated kynurenine levels have been found in humans in a number of diseases and after interferon treatment, and the enzyme is induced in rodents after administration of interferon inducers, or influenza virus. IDO induction also occurs in vivo during rejection of allogeneic tumors, indicating a possible role for this enzyme in the tumor rejection process. The gene for IDO has been cloned and shown to be differentially regulated by IFN-alpha and IFN-gamma. IDO induction has been correlated with induction of GTP-cyclohydrolase, the key enzyme in pteridine biosynthesis. A direct role for IDO in pteridine synthesis has not been shown, and this parallel induction may reflect coordinate regulation of genes induced by IFN-gamma. A possible role for IDO in O2-radical scavenging and in inflammation is discussed.     

Artemisinin: mechanisms of action,resistance and toxicity

Artemisinin and its derivatives are widely used throughout the world. The mechanism of action of these compounds appears to involve the heme-mediated decomposition of the endoperoxide bridge to produce carbon-centred free radicals. The involvement of heme explains why the drugs are selectively toxic to malaria parasites. The resulting carbon-centred free radicals are alkylate heme and proteins, one of which is the translationally controlled tumour protein. Clinically relevant artemisinin resistance has not been demonstrated, but it is likely to occur since artemisinin resistance has been obtained in laboratory models. At high doses, artemisinin can be neurotoxic but toxicity has not been found in clinical studies. The mechanism of neurotoxicity may be similar to the mechanism of action.     

Mechanisms of hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and inorganic pyrophosphate in aqueous perchloric acid

The acid-catalyzed hydrolysis of adenosine 5'-triphosphate (ATP) has been found to give rise both to adenosine 5'-diphosphate (ADP) and inorganic phosphate and to adenosine 5'-phosphate (AMP) and inorganic pyrophosphate. Kinetic and isotope studies on the mechanism of hydrolysis of ATP therefore depend on a knowledge of the mechanism of hydrolysis of the polyphosphate products, ADP and inorganic pyrophosphate. The latter reactions have been studied over the acidity range 1--5 M perchloric acid at 25 degrees C while the more complex problem of the hydrolysis of ATP has been followed at a single acidity (3 M perchloric acid). The positions of bond fission have been determined for both ATP and ADP.     

Structure,Function, and Mechanism of HhaI DNA Methyltransferases

A vast amount of literature has accumulated on the characterization of DNA methyltransferases. The HhaI DNA methyltransferase, a C5-cytosine methyltransferase, has been the subject of investigation for the last 2 decades. Biochemical and kinetic characterization have led to an understanding of the catalytic and kinetic mechanism of the methyltransfer reaction. The HhaI methyltransferase has also been subjected to extensive structural analysis, with the availability of 12 structures with or without a cofactor and a variety of DNA substrates. The mechanism of base flipping, first described for the HhaI methyltransferase, is conserved among all DNA methyltransferases and is also found to occur in numerous DNA repair enzymes. Studies with other methyltransferase reveal a significant structural and functional similarity among different types of methyltransferases. This review aims to summarize the available information on the HhaI DNA methyltransferase.     

Insights into SARS-CoV-2 genome,structure, evolution,pathogenesis and therapies: Structural genomics approach

The sudden emergence of severe respiratory disease, caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently become a public health emergency. Genome sequence analysis of SARS-CoV-2 revealed its close resemblance to the earlier reported SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). However, initial testing of the drugs used against SARS-CoV and MERS-CoV has been ineffective in controlling SARS-CoV-2. The present study highlights the genomic, proteomic, pathogenesis, and therapeutic strategies in SARS-CoV-2 infection. We have carried out sequence analysis of potential drug target proteins in SARS-CoV-2 and, compared them with SARS-CoV and MERS viruses. Analysis of mutations in the coding and non-coding regions, genetic diversity, and pathogenicity of SARS-CoV-2 has also been done. A detailed structural analysis of drug target proteins has been performed to gain insights into the mechanism of pathogenesis, structure-function relationships, and the development of structure-guided therapeutic approaches. The cytokine profiling and inflammatory signalling are different in the case of SARS-CoV-2 infection. We also highlighted possible therapies and their mechanism of action followed by clinical manifestation. Our analysis suggests a minimal variation in the genome sequence of SARS-CoV-2, may be responsible for a drastic change in the structures of target proteins, which makes available drugs ineffective.     

Inhibition of human dimethylarginine dimethylaminohydrolase-1 by S-nitroso-L-homocysteine and hydrogen peroxide. Analysis, quantification, and implications for hyperhomocysteinemia

The plasma concentrations of two cardiovascular risk factors, total homocysteine (tHcy) and asymmetric dimethylarginine (ADMA), correlate with decreased levels of endothelium-derived nitric oxide and subsequent endothelial dysfunction. Homocysteine has been proposed to inhibit the catabolic enzyme of ADMA, dimethylarginine dimethylaminohydrolase (DDAH), but the mechanism of this inhibition has not been fully elucidated. Here, the human DDAH isoform-1 (DDAH-1) is heterologously expressed and purified. Cys(274) and His(173) are identified as active site residues and the pH rate dependence is described. Because oxidation of the active site Cys has been suggested as an inhibitory mechanism in patients with hyperhomocysteinemia, the sensitivity of DDAH-1 to inhibition by L-homocysteine, H(2)O(2), and S-nitroso-L-homocysteine is quantified. DDAH-1 is surprisingly insensitive to inactivation by the powerful oxidant, H(2)O(2) (0.088 M(-1) s(-1)), possibly because of a substrate-assisted mechanism that allows the active site cysteine to remain predominantly protonated and less reactive in the resting enzyme. In contrast, DDAH-1 is sensitive to inactivation by S-nitroso-L-homocysteine (3.79 M(-1) s(-1)). This work illustrates how a particular catalytic mechanism can result in selective redox regulation and has possible implications for hyperhomocysteinemia.     

Kinetics of the inactivation of the protein-lipid complex, firefly luciferase, by sodium deoxycholate and its reactivation by phosphatidylcholine

Firefly luciferase has been shown to be a protein-lipid complex. Phospholipids and neutral lipids bound to luciferase have been identified. Sodium deoxycholate rapidly inactivated the enzyme, but an excess of phosphatidylcholine recovered luciferase activity. From the kinetics of inactivation and reactivation, a mechanism for interaction of the enzyme with detergents and phospholipids has been proposed. The substrates ATP and Mg2+ stabilized luciferase during delipidation.     

Cellular senescence, cancer, and organismal aging: a paradigm shift

Cellular senescence is an anti-cancer mechanism and may contribute to organismal aging. A change in paradigm has been proposed that cellular senescence may reduce cancer mortality rather than promote it late in life, and thus positively contributes to longevity in organisms with renewable tissues as a common mechanism across the species.     

Autophagy, mitochondrial quality control, and oncogenesis

Cancer is a disease associated with aging. More than 80% of human cancers are diagnosed in people aged 55 years or older. Autophagy has recently been demonstrated to be a novel mechanism of tumor suppression. Interestingly, autophagy also plays an important role in the control of aging. Here we summarize the genetic studies of autophagy in tumorigenesis and aging, and propose that autophagy may suppress tumor development and prevent aging through a common mechanism involving mitochondrial surveillance.     

Overexpression and mechanistic characterization of blastula protease 10, a metalloprotease involved in sea urchin embryogenesis and development

Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1. Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated "tyrosine switch." It has not been determined if the more structurally complex members of this family involved in eukaryotic development share this mechanism. The recombinant BP10 has been overexpressed in Escherichia coli, its metalloenzyme nature has been confirmed, and its catalytic properties have been characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native zinc-containing form and copper derivative. The copper derivative of BP10 shows a remarkable 960% rate acceleration toward the hydrolysis of the synthetic substrate N-benzoyl-arginine-p-nitroanilide when compared with the zinc form. The enzyme also shows calcium-dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-type enzymes in deuterostomes, which can add supporting data to corroborate the metal-centered mechanism proposed for astacin and the role of the coordinated Tyr. We have demonstrated the first mechanistic study of a tolloid-related metalloenzyme involved in sea urchin embryogenesis.     

Autophagy,Mitochondrial Quality Control,and Oncogenesis

Cancer is a disease associated with aging. More than 80% of human cancers are diagnosed in people aged 55 years or older. Autophagy has recently been demonstrated to be a novel mechanism of tumor suppression. Interestingly, autophagy also plays an important role in the control of aging. Here we summarize the genetic studies of autophagy in tumorigenesis and aging, and propose that autophagy may suppress tumor development and prevent aging through a common mechanism involving mitochondrial surveillance.     

Et tu, Grotthuss! and other unfinished stories

This review article is divided into three sections. In Section 1, a short biographical note on Freiherr von Grotthuss is followed by a detailed summary of the main findings and ideas present in his 1806 paper. Attempts to place Grotthuss contribution in the context of the science done at his time were also made. In Section 2, the modern version of the Grotthuss mechanism is reviewed. The classical Grotthuss model has been recently questioned and new mechanisms and ideas regarding proton transfer are briefly discussed. The last section discusses the significance of a classical Grotthuss mechanism for proton transfer in water chains inside protein cavities. This has been an interesting new twist in the ongoing history of the Grotthuss mechanism. A summary and discussion of what was learned from probably the simplest currently available experimental models of proton transfer in water wires in semi-synthetic ion channels are critically presented. This review ends discussing some of the questions that need to be addressed in the near future.     

Et tu, Grotthuss! and other unfinished stories

This review article is divided into three sections. In Section 1, a short biographical note on Freiherr von Grotthuss is followed by a detailed summary of the main findings and ideas present in his 1806 paper. Attempts to place Grotthuss contribution in the context of the science done at his time were also made. In Section 2, the modern version of the Grotthuss mechanism is reviewed. The classical Grotthuss model has been recently questioned and new mechanisms and ideas regarding proton transfer are briefly discussed. The last section discusses the significance of a classical Grotthuss mechanism for proton transfer in water chains inside protein cavities. This has been an interesting new twist in the ongoing history of the Grotthuss mechanism. A summary and discussion of what was learned from probably the simplest currently available experimental models of proton transfer in water wires in semi-synthetic ion channels are critically presented. This review ends discussing some of the questions that need to be addressed in the near future.     

Biosynthesis,biotechnological production,and application of teicoplanin: current state and perspectives

The glycopeptide teicoplanin isolated from the fermentation broth of Actinoplanes teichomyceticus is used to treat serious Gram-positive bacterial infections that are resistant to other antibiotics, e.g. β-lactams. The long time frame and progressively broader clinical use of teicoplanin has eventually led to the emergence and spreading of resistance in enterococci and staphylococci towards the antibiotics. Given the structural complexity of the natural product, only fermentative routes are available for bulk production of teicoplanin even though the total synthesis of the antibiotic has been accomplished. Because the low productivity (0.1–3.1 g/L) is a limitation to the commercial production of teicoplanin, substantial effort has been devoted to the strain improvement and process development for enhancing the productivity. This review summarizes the current state of the action mechanism, antibacterial activity, resistance mechanism, biotechnological production, and application of teicoplanin. Hyung-Moo Jung and Marimuthu Jeya equally contributed to this work.     

Death by design: apoptosis, necrosis and autophagy

Apoptosis is the principal mechanism by which cells are physiologically eliminated in metazoan organisms. During apoptotic death, cells are neatly carved up by caspases and packaged into apoptotic bodies as a mechanism to avoid immune activation. Recently, necrosis, once thought of as simply a passive, unorganized way to die, has emerged as an alternate form of programmed cell death whose activation might have important biological consequences, including the induction of an inflammatory response. Autophagy has also been suggested as a possible mechanism for non-apoptotic death despite evidence from many species that autophagy represents a survival strategy in times of stress. Recent advances have helped to define the function of and mechanism for programmed necrosis and the role of autophagy in cell survival and suicide.