Unique substrate specificity of ornithine aminotransferase from Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As an efficacious vaccine remains a challenge, chemotherapy is still the most effective way to combat the disease. In search of novel druggable targets, we performed a thorough characterization of the putative pyridoxal 5′-phosphate (PLP)-dependent enzyme ornithine aminotransferase from T. gondii ME49 (TgOAT). We overexpressed the protein in Escherichia coli and analysed its molecular and kinetic properties by UV-visible absorbance, fluorescence and CD spectroscopy, in addition to kinetic studies of both the steady state and pre-steady state. TgOAT is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity in vitro, suggesting a role in both arginine and GABA metabolism in vivo. The presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The findings that TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue make it an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention.

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dl-canaline and 5-fluoromethylornithine. Comparison of two inactivators of ornithine aminotransferase

Biochemical Journal ◽

10.1042/bj2680409 ◽

1990 ◽

Vol 268 (2) ◽

pp. 409-414 ◽

Cited By ~ 19

Author(s):

F N Bolkenius ◽

B Knödgen ◽

N Seiler

Keyword(s):

Rat Liver ◽

Active Site ◽

Absorption Maximum ◽

Intraperitoneal Administration ◽

Unsaturated Ketone ◽

Ornithine Aminotransferase ◽

Irreversible Inhibitor ◽

Oxime Formation

5-Fluoromethylornithine (5FMOrn) is an enzyme-activated irreversible inhibitor or ornithine aminotransferase (L-ornithine:2-oxo-acid 5-aminotransferase, OAT). For purified rat liver OAT, Ki(app.) was found to be 30 microM. and tau 1/2 = 4 min. Of the four stereomers of 5FMOrn only one reacts with OAT. The formation of a chromophore with an absorption maximum at 458 nm after inactivation of OAT by 5FMOrn suggests the formation of an enamine intermediate, which is slowly hydrolysed to release an unsaturated ketone. L-Canaline [(S)-2-amino-4-amino-oxybutyric acid] is a well-known irreversible inhibitor of OAT. Not only the natural L-enantiomer but also the D-enantiomer reacts by oxime formation with pyridoxal 5′-phosphate in the active site of the enzyme, although considerably more slowly. This demonstrates that the stereochemistry at C-2 of ornithine is not absolutely stringent. In vitro, canaline reacted faster than 5FMOrn with OAT. In vivo, however, only incomplete OAT inhibition was observed with canaline. Whereas intraperitoneal administration of 10 mg of 5FMOrn/kg body wt. to mice was sufficient to inactivate OAT in brain and liver by 90% for 24 h, 500 mg of DL-canaline/kg body wt. only produced a transient inhibition of 65-70%. The accumulation of ornithine in these tissues was considerably slower and the maximum concentrations lower than were achieved with 5FMOrn. It appears that DL-canaline, in contrast with 5FMOrn, is not useful as a tool in studies of biological consequences of OAT inhibition.

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Pre-Steady-State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-Family DNA Polymerases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01229-10 ◽

2010 ◽

Vol 55 (1) ◽

pp. 276-283 ◽

Cited By ~ 23

Author(s):

Jessica A. Brown ◽

Lindsey R. Pack ◽

Jason D. Fowler ◽

Zucai Suo

Keyword(s):

Mitochondrial Dna ◽

Steady State ◽

Substrate Specificity ◽

Dna Polymerase ◽

Dna Polymerases ◽

Steady State Kinetic ◽

Y Family ◽

State Kinetic

ABSTRACTNucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established bothin vitroandin vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypassin vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active, 5′-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5′-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.

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Three Kinetically Distinct Ca2+-Independent Depolarization-Activated K+ Currents in Callosal-Projecting Rat Visual Cortical Neurons

Journal of Neurophysiology ◽

10.1152/jn.1997.78.5.2309 ◽

1997 ◽

Vol 78 (5) ◽

pp. 2309-2320 ◽

Cited By ~ 23

Author(s):

Rachel E. Locke ◽

Jeanne M. Nerbonne

Keyword(s):

Steady State ◽

Cortical Neurons ◽

Differential Sensitivity ◽

Kinetic Properties ◽

Time Constants ◽

Steady State Inactivation ◽

Order Of Magnitude ◽

Visual Cortical

Locke, Rachel E. and Jeanne M. Nerbonne. Three kinetically distinct Ca2+-independent depolarization-activated K+ currents in callosal-projecting rat visual cortical neurons. J. Neurophysiol. 78: 2309–2320, 1997. Whole cell, Ca2+-independent, depolarization-activated K+ currents were characterized in identified callosal-projecting (CP) neurons isolated from postnatal day 7–16 rat primary visual cortex. CP neurons were identified in vitro after in vivo retrograde labeling with fluorescently tagged latex microbeads. During brief (160-ms) depolarizing voltage steps to potentials between −50 and +60 mV, outward K+ currents in these cells activate rapidly and inactivate to varying degrees. Three distinct K+ currents were separated based on differential sensitivity to 4-aminopyridine (4-AP); these are referred to here as I A, I D, and I K, because their properties are similar (but not identical) K+ currents termed I A, I D, and I K in other cells. The current sensitive to high (≥100 μM) concentrations of 4-AP ( I A) activates and inactivates rapidly; the current blocked completely by low (≤50 μM) 4-AP ( I D) activates rapidly and inactivates slowly. A slowly activating, slowly inactivating current ( I K) remains in the presence of 5 mM 4-AP. I A, I D, and I K also were separated and characterized in experiments that did not rely on the use of 4-AP. All CP cells express all three K+ current types, although the relative densities of I A, I D, and I K vary among cells. The experiments here also have revealed that I A, I D, and I K display similar voltage dependences of activation and steady state inactivation, whereas the kinetic properties of the currents are distinct. At +30 mV, for example, mean ± SD activation τs are 0.83 ± 0.24 ms for I A, 1.74 ± 0.49 ms for I D, and 14.7 ± 4.0 ms for I K. Mean ± SD inactivation τs for I A and I D are 26 ± 7 ms and 569 ± 143 ms, respectively. Inactivation of I K is biexponential with mean ± SD inactivation time constants of 475 ± 232 ms and 3,128 ± 1,328 ms; ∼20% of the 4-AP–insensitive current is noninactivating. For all three components, activation is voltage dependent, increasing with increasing depolarization, whereas inactivation is voltage independent. Both I A and I K recover rapidly from steady state inactivation with mean ± SD recovery time constants of 38 ± 7 ms and 79 ± 26 ms, respectively; I D recovers an order of magnitude more slowly (588 ± 274 ms). The properties of I A, I D, and I K in CP neurons are compared with those of similar currents described previously in other mammalian central neurons and, in the accompanying paper, the roles of these conductances in regulating the firing properties of CP neurons are explored.

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Oxidative stress as antifibrogenic stimulus? Low dose steady state H2O2 induces fibrolytic MMP–3 in vitro and in vivo

Zeitschrift für Gastroenterologie ◽

10.1055/s-0031-1295764 ◽

2012 ◽

Vol 50 (01) ◽

Author(s):

G Millonig ◽

S Ottinger ◽

HK Seitz ◽

S Mueller

Keyword(s):

Oxidative Stress ◽

Steady State ◽

Low Dose

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Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

Author(s):

Kathleen M. Donnelly ◽

Michael E. Bromberg ◽

Aaron Milstone ◽

Jennifer Madison McNiff ◽

Gordon Terwilliger ◽

...

Keyword(s):

Active Site ◽

Thrombin Generation ◽

Factor Xa ◽

Coagulation Factor ◽

Melanoma Cells ◽

Factor V ◽

Ancylostoma Caninum ◽

Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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Traceable Peptidic Ligands that Target Ghrelin Receptors

Current Protein and Peptide Science ◽

10.2174/1389203721666200702131457 ◽

2020 ◽

Vol 21 (10) ◽

pp. 955-964 ◽

Cited By ~ 1

Author(s):

Mengjie Liu ◽

John Wade ◽

Mohammed Akhter Hossain

Keyword(s):

In Vivo Imaging ◽

Peptide Hormone ◽

Structural Features ◽

Pharmacological Properties ◽

Imaging Studies ◽

Cognate Receptor ◽

Ghrelin Receptors ◽

Biochemical Research

: Ghrelin is a 28-amino acid octanoylated peptide hormone that is implicated in many physiological and pathophysiological processes. Specific visualization of ghrelin and its cognate receptor using traceable ligands is crucial in elucidating the localization, functions, and expression pattern of the peptide’s signaling pathway. Here 12 representative radio- and fluorescently-labeled peptide-based ligands are reviewed for in vitro and in vivo imaging studies. In particular, the focus is on their structural features, pharmacological properties, and applications in further biochemical research.

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Polyamines and Related Nitrogen Compounds in the Chemotherapy of Neglected Diseases Caused by Kinetoplastids

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666180427151338 ◽

2018 ◽

Vol 18 (5) ◽

pp. 321-368 ◽

Cited By ~ 2

Author(s):

Juan A. Bisceglia ◽

Maria C. Mollo ◽

Nadia Gruber ◽

Liliana R. Orelli

Keyword(s):

Drug Targets ◽

Redox Homeostasis ◽

Cost Effective ◽

Structural Features ◽

Mammalian Host ◽

Neglected Diseases ◽

Nitrogen Compounds ◽

Chemical Structures

Neglected diseases due to the parasitic protozoa Leishmania and Trypanosoma (kinetoplastids) affect millions of people worldwide, and the lack of suitable treatments has promoted an ongoing drug discovery effort to identify novel nontoxic and cost-effective chemotherapies. Polyamines are ubiquitous small organic molecules that play key roles in kinetoplastid parasites metabolism, redox homeostasis and in the normal progression of cell cycles, which differ from those found in the mammalian host. These features make polyamines attractive in terms of antiparasitic drug development. The present work provides a comprehensive insight on the use of polyamine derivatives and related nitrogen compounds in the chemotherapy of kinetoplastid diseases. The amount of literature on this subject is considerable, and a classification considering drug targets and chemical structures were made. Polyamines, aminoalcohols and basic heterocycles designed to target the relevant parasitic enzyme trypanothione reductase are discussed in the first section, followed by compounds directed to less common targets, like parasite SOD and the aminopurine P2 transporter. Finally, the third section comprises nitrogen compounds structurally derived from antimalaric agents. References on the chemical synthesis of the selected compounds are reported together with their in vivo and/or in vitro IC50 values, and structureactivity relationships within each group are analyzed. Some favourable structural features were identified from the SAR analyses comprising protonable sites, hydrophobic groups and optimum distances between them. The importance of certain pharmacophoric groups or amino acid residues in the bioactivity of polyamine derived compounds is also discussed.

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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Interaction of Lactoferrin with Unsaturated Fatty Acids: In Vitro and In Vivo Study of Human Lactoferrin/Oleic Acid Complex Cytotoxicity

Materials ◽

10.3390/ma14071602 ◽

2021 ◽

Vol 14 (7) ◽

pp. 1602

Author(s):

Anna Elizarova ◽

Alexey Sokolov ◽

Valeria Kostevich ◽

Ekaterina Kisseleva ◽

Evgeny Zelenskiy ◽

...

Keyword(s):

Oleic Acid ◽

Structural Changes ◽

Unsaturated Fatty Acids ◽

Structural Features ◽

Control Group ◽

Acid Complex ◽

Hepatoma 22A ◽

Constitutive Parameters

As shown recently, oleic acid (OA) in complex with lactoferrin (LF) causes the death of cancer cells, but no mechanism(s) of that toxicity have been disclosed. In this study, constitutive parameters of the antitumor effect of LF/OA complex were explored. Complex LF/OA was prepared by titrating recombinant human LF with OA. Spectral analysis was used to assess possible structural changes of LF within its complex with OA. Structural features of apo-LF did not change within the complex LF:OA = 1:8, which was toxic for hepatoma 22a cells. Cytotoxicity of the complex LF:OA = 1:8 was tested in cultured hepatoma 22a cells and in fresh erythrocytes. Its anticancer activity was tested in mice carrying hepatoma 22a. In mice injected daily with LF-8OA, the same tumor grew significantly slower. In 20% of animals, the tumors completely resolved. LF alone was less efficient, i.e., the tumor growth index was 0.14 for LF-8OA and 0.63 for LF as compared with 1.0 in the control animals. The results of testing from 48 days after the tumor inoculation showed that the survival rate among LF-8OA-treated animals was 70%, contrary to 0% rate in the control group and among the LF-treated mice. Our data allow us to regard the complex of LF and OA as a promising tool for cancer treatment.

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