scholarly journals The Use Haloperoxidases in Organic Synthesis: Selected Reactions of Oxidation, Epoxydation and Sulfoxidation

2017 ◽  
Vol 4 (4) ◽  
pp. 221 ◽  
Author(s):  
V.M. Dembitsky ◽  
M. Srebnik
Keyword(s):  
Hydrogen Peroxide ◽  
Substrate Specificity ◽  
Organic Synthesis ◽  
Active Site ◽  
Organic Molecules ◽  
Molecular Genetic ◽  
Metabolite Formation ◽  
Wide Range ◽  
Mechanism Of Oxidation

<p>Haloperoxidases are ubiquitous metalloenzymes that catalyse a variety of enantioselective oxygen-transfer reactions with hydrogen peroxide or alkylperoxides. Haloperoxidases are enzymes which catalyze the reaction of oxidation, epoxidation and sulfoxidation by hydrogen peroxide. These enzymes usually contain the FeHeme moiety or vanadium as an essential constituent at their active site, however, a few haloperoxidases which lack a metal cofactor are known. This review will examine the reactivity of the different haloperoxidases, particularly the mechanism of oxidation by hydrogen peroxide, and the mechanism of oxidation and sulfoxidation, including the newly reported regioselectivity and enantioselectivity of the haloperoxidases. The structure of chloroperoxidase, the vanadium active site and the role of critical amino acid side chains for catalysis and functional biomimetic systems, with specific relevance to the mechanism of the haloperoxidase enzymes. Advances have recently been made in using them to prepare, under controlled conditions, chiral organic molecules that are valuable for the synthesis of a wide range of useful compounds. The application of biocatalytic methods in asymmetric organic synthesis is of great interest as an alternative to chemical procedures employing chiral auxiliaries. Asymmetric oxidation of prochiral sulfides to yield optically active sulfoxides has been performed by many different techniques yielding varying enantiomeric excess values. Oxygenated metabolites are compounds that are commonly found in nature and they are produced by many different organisms. The oxygen atom is incorporated into organic compounds by enzyme-catalyzed reactions with oxygen ions as the oxygen source. For over 40 years haloperoxidases were thought to be responsible for the incorporation of mainly halogen atoms into organic molecules. However, haloperoxidases lack substrate specificity and regioselectivity, and the connection of haloperoxidases with the in vivo formation of oxygenated as well as halometabolites has been demonstrated. Recently, molecular genetic investigations showed that, at least in bacteria, fungi, and other organisms a different class of halogenases is involved in halo- and oxygenated metabolite formation. These halogenases were found to require FADH2, which can be produced from FAD and NADH by unspecific flavin reductases. The FADH<sub>2</sub>-dependent halogenases and haloperoxidases show substrate specificity and regioselectivity, and their genes have been detected in many halometabolite-producing organisms, suggesting that this type of halogenating enzymes constitutes the major source for halo- and oxygenated metabolite formation in bacteria and also in other organisms. Distribution of haloperoxidases in nature also is demonstrated in this brief review.</p>

scholarly journals Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I

2020 ◽  
Vol 21 (16) ◽  
pp. 5734
Author(s):  
Joaquin Ramirez-Ramirez ◽  
Javier Martin-Diaz ◽  
Nina Pastor ◽  
Miguel Alcalde ◽  
Marcela Ayala
Keyword(s):  
Hydrogen Peroxide ◽  
Cytochrome P450 ◽  
Active Site ◽  
Bulk Solution ◽  
Computational Studies ◽  
Organic Substrates ◽  
Oxidation Reactions ◽  
Heme Group ◽  
Wide Range

Unspecific peroxygenases (UPOs) are fungal heme-thiolate enzymes able to catalyze a wide range of oxidation reactions, such as peroxidase-like, catalase-like, haloperoxidase-like, and, most interestingly, cytochrome P450-like. One of the most outstanding properties of these enzymes is the ability to catalyze the oxidation a wide range of organic substrates (both aromatic and aliphatic) through cytochrome P450-like reactions (the so-called peroxygenase activity), which involves the insertion of an oxygen atom from hydrogen peroxide. To catalyze this reaction, the substrate must access a channel connecting the bulk solution to the heme group. The composition, shape, and flexibility of this channel surely modulate the catalytic ability of the enzymes in this family. In order to gain an understanding of the role of the residues comprising the channel, mutants derived from PaDa-I, a laboratory-evolved UPO variant from Agrocybe aegerita, were obtained. The two phenylalanine residues at the surface of the channel, which regulate the traffic towards the heme active site, were mutated by less bulky residues (alanine and leucine). The mutants were experimentally characterized, and computational studies (i.e., molecular dynamics (MD)) were performed. The results suggest that these residues are necessary to reduce the flexibility of the region and maintain the topography of the channel.

scholarly journals Effect of Sequences of the Active-Site Dipeptides of DsbA and DsbC on In Vivo Folding of Multidisulfide Proteins inEscherichia coli

2001 ◽  
Vol 183 (3) ◽  
pp. 980-988 ◽  
Author(s):  
Paul H. Bessette ◽  
Ji Qiu ◽  
James C. A. Bardwell ◽  
James R. Swartz ◽  
George Georgiou
Keyword(s):  
Redox Potential ◽  
Active Site ◽  
Active Sites ◽  
Multicopy Plasmid ◽  
Redox Potentials ◽  
Cxxc Motif ◽  
Wide Range ◽  
Significant Difference ◽  
Disulfide Isomerization

ABSTRACT We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. ThedsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.

scholarly journals Demonstration of Redox Potential ofMetschnikowia koreensisfor Stereoinversion of Secondary Alcohols/1,2-Diols

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Vachan Singh Meena ◽  
Linga Banoth ◽  
U. C. Banerjee
Keyword(s):  
Substrate Specificity ◽  
Redox Potential ◽  
Optical Purity ◽  
Cofactor Regeneration ◽  
High Yield ◽  
Secondary Alcohols ◽  
One Pot ◽  
Wide Range ◽  
Multistep Reaction

The present work reports theMetschnikowia koreensis-catalyzed one-pot deracemization of secondary alcohols/1,2-diols and their derivatives with in vivo cofactor regeneration. Reaction is stereoselective and proceeds with sequential oxidation of (R)-secondary alcohols to the corresponding ketones and the reduction of the ketones to (S)-secondary alcohols. Method is applicable to a repertoire of racemic aryl secondary alcohols and 1,2-diols establishing a wide range of substrate specificity ofM. koreensis. This ecofriendly method afforded the product in high yield (88%) and excellent optical purity (>98%ee), minimizing the requirement of multistep reaction and expensive cofactor.

scholarly journals Molecular Mechanisms of Substrate Recognition and Specificity of New Delhi Metallo-β-Lactamase

2014 ◽  
Vol 58 (9) ◽  
pp. 5372-5378 ◽  
Author(s):  
Jiachi Chiou ◽  
Thomas Yun-Chung Leung ◽  
Sheng Chen
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Substrate Recognition ◽  
New Delhi ◽  
Structure Comparison ◽  
Content Type ◽  
Crystallographic Study ◽  
Wide Range ◽  
Active Site Cavity

ABSTRACTCarbapenems are one of the last lines of defense for Gram-negative pathogens, such as members of theEnterobacteriaceae. Despite the fact that most carbapenems are resistant to extended-spectrum β-lactamase (ESBL), emerging metallo-β-lactamases (MBLs), including New Delhi metallo-β-lactamase 1 (NDM-1), that can hydrolyze carbapenems have become prevalent and are frequently associated with the so-called “superbugs,” for which treatments are extremely limited. Crystallographic study sheds light on the modes of antibiotic binding to NDM-1, yet the mechanisms governing substrate recognition and specificity are largely unclear. This study provides a connection between crystallographic study and the functional significance of NDM-1, with an emphasis on the substrate specificity and catalysis of various β-lactams. L1 loop residues L59, V67, and W87were important for the activity of NDM-1, most likely through maintaining the partial folding of the L1 loop or active site conformation through hydrophobic interaction with the R groups of β-lactams or the β-lactam ring. Substitution of alanine for L59showed greater reduction of MICs to ampicillin and selected cephalosporins, whereas substitutions of alanine for V67had more impact on the MICs of carbapenems. K224and N233on the L3 loop played important roles in the recognition of substrate and contributed to substrate hydrolysis. These data together with the structure comparison of the B1 and B2 subclasses of MBLs revealed that the broad substrate specificity of NDM-1 could be due to the ability of its wide active site cavity to accommodate a wide range of β-lactams. This study provides insights into the development of efficient inhibitors for NDM-1 and offers an efficient tactic with which to study the substrate specificities of other β-lactamases.

scholarly journals Structure-function analyses of alkylhydroperoxidase D from Streptococcus pneumoniae reveal an unusual three-cysteine active site architecture

2020 ◽  
Vol 295 (10) ◽  
pp. 2984-2999 ◽  
Author(s):  
Yanxiang Meng ◽  
Campbell R. Sheen ◽  
Nicholas J. Magon ◽  
Mark B. Hampton ◽  
Renwick C. J. Dobson
Keyword(s):  
Hydrogen Peroxide ◽  
Electron Transfer ◽  
Streptococcus Pneumoniae ◽  
Stress Responses ◽  
Active Site ◽  
Quaternary Structure ◽  
Cysteine Oxidation ◽  
Core Motif ◽  
Mediate Electron Transfer

During aerobic growth, the Gram-positive facultative anaerobe and opportunistic human pathogen Streptococcus pneumoniae generates large amounts of hydrogen peroxide that can accumulate to millimolar concentrations. The mechanism by which this catalase-negative bacterium can withstand endogenous hydrogen peroxide is incompletely understood. The enzyme alkylhydroperoxidase D (AhpD) has been shown to contribute to pneumococcal virulence and oxidative stress responses in vivo. We demonstrate here that SpAhpD exhibits weak thiol-dependent peroxidase activity and, unlike the previously reported Mycobacterium tuberculosis AhpC/D system, SpAhpD does not mediate electron transfer to SpAhpC. A 2.3-Å resolution crystal structure revealed several unusual structural features, including a three-cysteine active site architecture that is buried in a deep pocket, in contrast to the two-cysteine active site found in other AhpD enzymes. All single-cysteine SpAhpD variants remained partially active, and LC-MS/MS analyses revealed that the third cysteine, Cys-163, formed disulfide bonds with either of two cysteines in the canonical Cys-78-X–X-Cys-81 motif. We observed that SpAhpD formed a dimeric quaternary structure both in the crystal and in solution, and that the highly conserved Asn-76 of the AhpD core motif is important for SpAhpD folding. In summary, SpAhpD is a weak peroxidase and does not transfer electrons to AhpC, and therefore does not fit existing models of bacterial AhpD antioxidant defense mechanisms. We propose that it is unlikely that SpAhpD removes peroxides either directly or via AhpC, and that SpAhpD cysteine oxidation may act as a redox switch or mediate electron transfer with other thiol proteins.

Unique substrate specificity of ornithine aminotransferase from Toxoplasma gondii

2017 ◽  
Vol 474 (6) ◽  
pp. 939-955 ◽  
Author(s):  
Alessandra Astegno ◽  
Elena Maresi ◽  
Mariarita Bertoldi ◽  
Valentina La Verde ◽  
Alessandro Paiardini ◽  
...  
Keyword(s):  
Steady State ◽  
Toxoplasma Gondii ◽  
Substrate Specificity ◽  
Active Site ◽  
Structural Features ◽  
Kinetic Properties ◽  
Ornithine Aminotransferase ◽  
Human Homologue

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.

scholarly journals Redox signaling modulates Rho activity and tissue contractility in the C. elegans spermatheca

2019 ◽  
Author(s):  
Charlotte A. Kelley ◽  
Sasha De Henau ◽  
Liam Bell ◽  
Tobias B. Dansen ◽  
Erin J. Cram
Keyword(s):  
Hydrogen Peroxide ◽  
Active Site ◽  
Myoepithelial Cells ◽  
Redox Signaling ◽  
Small Gtpase ◽  
C Elegans ◽  
Hydrogen Peroxide Treatment ◽  
Fine Tune ◽  
Contractile Tissue

AbstractActomyosin based contractility in smooth muscle and non-muscle cells is regulated by signaling through the small GTPase Rho and by calcium-activated pathways. We use the myoepithelial cells of the Caenorhabditis elegans spermatheca to study the mechanisms of coordinated myosin activation in vivo. Here, we demonstrate that redox signaling regulates RHO-1/Rho activity in this contractile tissue. Exogenous hydrogen peroxide treatment decreases spermathecal contractility by inhibiting RHO-1, which is mediated through a conserved cysteine in its active site (C20). Further, we identify a gradient of oxidation across the spermathecal tissue, which is regulated by the cytosolic superoxide dismutase, SOD-1. SOD-1 functions in the Rho pathway to inhibit RHO-1 through oxidation of C20. Our results suggest that SOD-1 functions to regulate the redox environment and to fine-tune Rho activity across the spermatheca.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light &gt;600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
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.

Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan
Keyword(s):  
Soft Tissue ◽  
Room Temperature ◽  
Normal Subjects ◽  
Left Testis ◽  
Wide Range ◽  
Activity Ratios ◽  
The Right ◽  
Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

Sign in / Sign up

Export Citation Format

Share Document