scholarly journals Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

2020 ◽  
Author(s):  
Laine Lysyk ◽  
Raelynn Brassard ◽  
Elena Arutyunova ◽  
Verena Siebert ◽  
Zhenze Jiang ◽  
...  
Keyword(s):  
Catalytic Properties ◽  
Side Chain ◽  
Turnover Rates ◽  
Rhomboid Protease ◽  
Mitochondrial Homeostasis ◽  
Substrate Preferences ◽  
Rhomboid Proteases ◽  
Hydrolysis Of ◽  
Insight Into

AbstractThe rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac, which have crucial roles in mitochondrial quality control and apoptosis. To gain insight into the catalytic properties of the PARL protease, we expressed human PARL in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show PARL activity in detergent is enhanced by cardiolipin. Significantly higher turnover rates are observed for PARL reconstituted in proteoliposomes, with Smac being cleaved most rapidly at a rate of 1 min−1. PGAM5 is cleaved with the highest efficiency compared to PINK1 and Smac. In proteoliposomes, a truncated β-cleavage form of PARL is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5 and Smac. Multiplex substrate profiling reveals a substrate preference for PARL with a bulky side chain Phe in P1, which is distinct from small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences.

scholarly journals Acyl-CoA oxidase complexes control the chemical message produced by Caenorhabditis elegans

2015 ◽  
Vol 112 (13) ◽  
pp. 3955-3960 ◽  
Author(s):  
Xinxing Zhang ◽  
Likui Feng ◽  
Satya Chinta ◽  
Prashant Singh ◽  
Yuting Wang ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Environmental Conditions ◽  
Side Chain ◽  
Side Chains ◽  
Activity Assay ◽  
C Elegans ◽  
Substrate Preferences ◽  
Acyl Coa Oxidase ◽  
Coa Thioesters

Caenorhabditis elegans uses ascaroside pheromones to induce development of the stress-resistant dauer larval stage and to coordinate various behaviors. Peroxisomal β-oxidation cycles are required for the biosynthesis of the fatty acid-derived side chains of the ascarosides. Here we show that three acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, form different protein homo- and heterodimers with distinct substrate preferences. Mutations in the acyl-CoA oxidase genes acox-1, -2, and -3 led to specific defects in ascaroside production. When the acyl-CoA oxidases were expressed alone or in pairs and purified, the resulting acyl-CoA oxidase homo- and heterodimers displayed different side-chain length preferences in an in vitro activity assay. Specifically, an ACOX-1 homodimer controls the production of ascarosides with side chains with nine or fewer carbons, an ACOX-1/ACOX-3 heterodimer controls the production of those with side chains with seven or fewer carbons, and an ACOX-2 homodimer controls the production of those with ω-side chains with less than five carbons. Our results support a biosynthetic model in which β-oxidation enzymes act directly on the CoA-thioesters of ascaroside biosynthetic precursors. Furthermore, we identify environmental conditions, including high temperature and low food availability, that induce the expression of acox-2 and/or acox-3 and lead to corresponding changes in ascaroside production. Thus, our work uncovers an important mechanism by which C. elegans increases the production of the most potent dauer pheromones, those with the shortest side chains, under specific environmental conditions.

scholarly journals Structure of a Talaromyces pinophilus GH62 arabinofuranosidase in complex with AraDNJ at 1.25 Å resolution

2018 ◽  
Vol 74 (8) ◽  
pp. 490-495 ◽  
Author(s):  
Olga V. Moroz ◽  
Lukasz F. Sobala ◽  
Elena Blagova ◽  
Travis Coyle ◽  
Wei Peng ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulosic Biomass ◽  
Structural Basis ◽  
Side Chain ◽  
Polymeric Substrates ◽  
Hydrolysis Of ◽  
Insight Into ◽  
Positively Charged ◽  
Talaromyces Pinophilus ◽  
Resolution Structure

The enzymatic hydrolysis of complex plant biomass is a major societal goal of the 21st century in order to deliver renewable energy from nonpetroleum and nonfood sources. One of the major problems in many industrial processes, including the production of second-generation biofuels from lignocellulose, is the presence of `hemicelluloses' such as xylans which block access to the cellulosic biomass. Xylans, with a polymeric β-1,4-xylose backbone, are frequently decorated with acetyl, glucuronyl and arabinofuranosyl `side-chain' substituents, all of which need to be removed for complete degradation of the xylan. As such, there is interest in side-chain-cleaving enzymes and their action on polymeric substrates. Here, the 1.25 Å resolution structure of the Talaromyces pinophilus arabinofuranosidase in complex with the inhibitor AraDNJ, which binds with a K d of 24 ± 0.4 µM, is reported. Positively charged iminosugars are generally considered to be potent inhibitors of retaining glycosidases by virtue of their ability to interact with both acid/base and nucleophilic carboxylates. Here, AraDNJ shows good inhibition of an inverting enzyme, allowing further insight into the structural basis for arabinoxylan recognition and degradation.

scholarly journals New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

2013 ◽  
Vol 79 (11) ◽  
pp. 3336-3345 ◽  
Author(s):  
Jinsol Heo ◽  
Se Hyeuk Kim ◽  
Pyung Cheon Lee
Keyword(s):  
Carotenoid Cleavage Dioxygenase ◽  
Cleavage Reaction ◽  
Natural Structure ◽  
Content Type ◽  
Substrate Preferences ◽  
Cleavage Reactions ◽  
Pcc 7120 ◽  
Insight Into

ABSTRACTCarotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD ofNostocsp. strain PCC 7120,in vivoandin vitrousing natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDsin vivoandin vitro.

scholarly journals Biochemical and Molecular Characterization of Three New Variants of AmpC β-Lactamases from Morganella morganii

2006 ◽  
Vol 50 (3) ◽  
pp. 962-967 ◽  
Author(s):  
Pablo Power ◽  
Moreno Galleni ◽  
Juan A. Ayala ◽  
Gabriel Gutkind
Keyword(s):  
Amino Acid ◽  
Catalytic Properties ◽  
Amino Acid Sequences ◽  
Major Influence ◽  
Morganella Morganii ◽  
Encoding Gene ◽  
Hydrolysis Of ◽  
Similar Kinetic

ABSTRACT Morganella morganii produces an inducible, chromosomally encoded AmpC β-lactamase. We describe in this study three new variants of AmpC within this species with apparent pIs of 6.6 (M19 from M. morganii strain PP19), 7.4 (M29 from M. morganii strain PP29), and 7.8 (M37 from M. morganii strain PP37). After gene sequencing, deduced amino acid sequences displayed one to six substitutions when compared to the available Morganella AmpC sequences. An AmpR-encoding gene was also found upstream of ampC, including the LysR regulators' helix-turn-helix DNA-binding domain and the putative T-N11-A-protected region in the ampR-ampC intercistronic sequence. All three AmpC variants were purified from in vitro-generated derepressed mutants and showed overall similar kinetic parameters. None of the observed amino acid changes, occurring at the surface of the protein, appear to have a major influence in their catalytic properties. Morganella AmpCs exhibit the highest catalytic efficiencies (kcat /Km ) on classical penicillins, cefoxitin, narrow-spectrum cephalosporins, and cefotaxime. Cefotaxime was more effectively hydrolyzed than other oxyimino-cephalosporins, whereas cefepime was 3 log-fold less efficiently hydrolyzed than other cephalosporins such as cephalothin. Several differences with other AmpC β-lactamases were found. Ampicillin was more efficiently hydrolyzed than benzylpenicillin. High kcat /Km values were observed for oxacillin and piperacillin, which are usually poor substrates for AmpC. A fairly efficient hydrolysis of imipenem was detected as well. Aztreonam, carbenicillin, and tazobactam were effective transient inactivators of these variants.

scholarly journals Impaired Inhibition by Avibactam and Resistance to the Ceftazidime-Avibactam Combination Due to the D179Y Substitution in the KPC-2 β-Lactamase

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Fabrice Compain ◽  
Michel Arthur
Keyword(s):  
Catalytic Properties ◽  
Residual Activity ◽  
Underlying Mechanism ◽  
Content Type ◽  
Y Substitution ◽  
Therapeutic Alternatives ◽  
Emergence Of Resistance ◽  
Hydrolysis Of ◽  
Insight Into

ABSTRACT The ceftazidime-avibactam antibiotic combination was recently shown to be at risk for the emergence of resistance under treatment. To gain insight into the underlying mechanism, we have analyzed the catalytic properties of a Klebsiella pneumoniae carbapenemase type 2 (KPC-2) β-lactamase harboring the D179Y substitution. We show that impaired inhibition by avibactam combined with significant residual activity for ceftazidime hydrolysis accounts for the resistance. In contrast, the D179Y substitution abolished the hydrolysis of aztreonam and imipenem, indicating that these drugs might provide therapeutic alternatives.

Synthesis and In Vitro Biological Evaluation of β,γ-Methano Analogues of Methotrexate and Aminopterin

Pteridines ◽  
1990 ◽  
Vol 2 (3) ◽  
pp. 133-139 ◽  
Author(s):  
Andre Rosowsky ◽  
Ronald A. Forsch ◽  
Richard G. Moran ◽  
James H. Freisheim
Keyword(s):  
Alkaline Hydrolysis ◽  
Cyclopropane Ring ◽  
Biological Evaluation ◽  
Side Chain ◽  
Activity Data ◽  
Folylpolyglutamate Synthetase ◽  
Conformationally Restricted ◽  
Diastereomer Mixture ◽  
Hydrolysis Of

Summaryβ,γ-Methano derivatives of methotrexate (MTX) and aminopterin (AMT) were synthesized with the aim of assessing the effect of this side-chain modification on dihydrofolate reductase (OHFR) inhibition, folylpolyglutamate synthetase (FPGS) inhibition, and tumor cell growth inhibition. Mixed carboxylic-carbonic anhydride (MCA) coupling of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and dimethyl trans-α-(2-carboxycyclopropyl) glycinate, followed by alkaline hydrolysis, afforded N-( 4-amino-4-deoxy-N10-methylpteroyl)-α-( trans-2-carboxycyclopropyl)glycine (β,γ-methanoMTX) as a mixture of the four possible diastereomers with trans substitution on the cyclopropane ring. N-(4-Amino-4-deoxypteroyl)-trans-α.-(2-carboxycyclopropyl) glycine (β,γ-methanoAMT) , also as a diastereomer mixture, was obtained from 4-amino-4-deoxy-N10 - formylpteroic acid (fAPA) and dimethyl trans-α-(2-carboxycyclopropyl)-glycinate by MCA coupling and alkaline hydrolysis of the ester and N10-formyl groups. β,γ-MethanoMTX and β,γ-methanoAMT may be viewed as MTX and AMT analogues with a conformationally restricted side chain. In vitro biological activity data for these novel compounds support the view that the active site of DHFR, already known for its ability to tolerate modification of the γ-carboxyl group of MTX and AMT, can likewise accommodate substitution on the β- and γ-carbons.

scholarly journals In Vitro and In Vivo Activities of Syn2190, a Novel β-Lactamase Inhibitor

1999 ◽  
Vol 43 (8) ◽  
pp. 1895-1900 ◽  
Author(s):  
Kouichi Nishida ◽  
Chieko Kunugita ◽  
Tatsuya Uji ◽  
Fusahiro Higashitani ◽  
Akio Hyodo ◽  
...  
Keyword(s):  
Systemic Infection ◽  
Side Chain ◽  
Infection Model ◽  
Morganella Morganii ◽  
Infection Models ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Group 1

ABSTRACT Syn2190, a monobactam derivative containing 1,5-dihydroxy-4-pyridone as the C-3 side chain, is a potent inhibitor of group 1 β-lactamase. The concentrations of inhibitor needed to reduce the initial rate of hydrolysis of substrate by 50% for Syn2190 against these enzymes were in the range of 0.002 to 0.01 μM. These values were 220- to 850-fold lower than those of tazobactam. Syn2190 showed in vitro synergy with ceftazidime and cefpirome. This synergy was dependent on the concentration of the inhibitor against group 1 β-lactamase-producing strains, such as Pseudomonas aeruginosa, Enterobacter cloacae, Citrobacter freundii, and Morganella morganii. However, against β-lactamase-derepressed mutants of P. aeruginosa, the MICs of ceftazidime plus Syn2190 were not affected by the amount of β-lactamase, and the values were the same for the parent strains. The MICs at which 50% of isolates are inhibited (MIC50s) of ceftazidime plus Syn2190 were 2- to 16-fold lower than those of ceftazidime alone for ceftazidime-resistant, clinically isolated gram-negative bacteria. Similarly, the MIC50s of cefpirome plus Syn2190 were two- to eightfold lower for cefpirome-resistant clinical isolates. The synergies of Syn2190 plus ceftazidime or cefpirome observed in vitro were also reflected in vivo. Syn2190 improved the efficacies of both cephalosporins in both a murine systemic infection model with cephalosporin-resistant rods and urinary tract infection models with cephalosporin-resistant P. aeruginosa.

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

2017 ◽  
Vol 73 (12) ◽  
pp. 970-984 ◽  
Author(s):  
Shenyuan Xu ◽  
Brian N. Long ◽  
Gabriel H. Boris ◽  
Anqi Chen ◽  
Shuisong Ni ◽  
...  
Keyword(s):  
Transition State ◽  
Binding Pocket ◽  
High Energy ◽  
Side Chain ◽  
Conformation Change ◽  
Gtp Hydrolysis ◽  
Structural Insight ◽  
Hydrolysis Of ◽  
Insight Into ◽  
Intrinsic Gtpase Activity

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

scholarly journals A rhomboid protease, EhROM1, regulates the submembrane distribution and size of the Gal/GalNAc lectin subunits in the human protozoan parasite, Entamoeba histolytica

2019 ◽  
Author(s):  
Brenda H. Welter ◽  
Lesly A. Temesvari
Keyword(s):  
Cell Surface ◽  
Entamoeba Histolytica ◽  
Molecular Mechanisms ◽  
Mutant Cell ◽  
Protozoan Parasite ◽  
Rhomboid Protease ◽  
Host Interactions ◽  
Rhomboid Proteases ◽  
Lipid Environment ◽  
Insight Into

AbstractEntamoeba histolytica is a food- and waterborne parasite that is the causative agent of amebic dysentery and amoebic liver abscesses. Adhesion is one of the most important virulence functions as it facilitates motility, colonization of host, destruction of host tissue, and uptake of nutrients by the parasite. One well-characterized parasite cell surface adhesin is the Gal/GalNAc lectin, which binds to galactose or N-acetylgalactosamine residues on host components and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Igl has been shown to be constitutively localized to lipid rafts (cholesterol-rich membrane domains), whereas Hgl and Lgl transiently associate with rafts. When all three subunits are localized to rafts there is an increase in galactose-sensitive adhesion. Thus, submembrane location may regulate the function of this adhesion. Rhomboid proteases are a conserved family of intramembrane proteases that also participate in the regulation of parasite-host interactions. In E. histolytica, one rhomboid protease, EhROM1, cleaves Hgl as a substrate, and knockdown of its expression inhibits parasite-host interactions. Since rhomboid proteases are found within membranes, it is not surprising that lipid composition regulates their activity and enzyme-substrate binding. Given the importance of the lipid environment for both rhomboid proteases and the Gal/GalNAc lectin, we sought to gain insight into the relationship between rhomboid proteases and submembrane location of the lectin in E. histolytica. We demonstrated that EhROM1, itself, is enriched in rafts. Reducing rhomboid protease activity, either pharmacologically or genetically, correlated with an enrichment of Hgl and Lgl in rafts. In a mutant cell line with reduced EhROM1 expression, there was also a significant augmentation of the level of all three Gal/GalNAc subunits on the cell surface and an increase in the molecular weight of Hgl and Lgl. Overall, the study provides insight into the molecular mechanisms governing parasite-host adhesion for this pathogen.

scholarly journals Non-Specific GH30_7 Endo-β-1,4-xylanase from Talaromyces leycettanus

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4614
Author(s):  
Katarína Šuchová ◽  
Nikolaj Spodsberg ◽  
Kristian B. R. Mørkeberg Krogh ◽  
Peter Biely ◽  
Vladimír Puchart
Keyword(s):  
General Formula ◽  
Main Chain ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Side Chain ◽  
Biotechnological Applications ◽  
Xylose Residue ◽  
Great Tolerance ◽  
Chain Cleavage ◽  
Hydrolysis Of

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-β-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear β-1,3-β-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched β-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear β-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the −2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.

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