TY - JOUR AU - Hajdú, István AU - Szilágyi, András AU - Végh, Barbara AU - Wacha, András Ferenc AU - Györffy, Dániel AU - Gráczer, Éva Laura AU - Somogyi, Márk AU - Gál, Péter AU - Závodszky, Péter TI - Ligand-induced conformational rearrangements regulate the switch between membrane-proximal and distal functions of Rho kinase 2. JF - COMMUNICATIONS BIOLOGY J2 - COMMUN BIOL VL - 3 PY - 2020 IS - 1 SN - 2399-3642 DO - 10.1038/s42003-020-01450-x UR - https://m2.mtmt.hu/api/publication/31677587 ID - 31677587 N1 - Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary ELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Institute of Biology, ELTE Eötvös Loránd University, Budapest, Hungary Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Budapest, Hungary Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary Export Date: 16 December 2020 Correspondence Address: Hajdú, I.; Institute of Enzymology, Research Centre for Natural Sciences, Institute of Enzymology, Research Centre for Natural SciencesHungary; email: hajdu.istvan@ttk.hu Funding details: National Research, Development and Innovation Office Funding details: Nemzeti Kutatási Fejlesztési és Innovációs Hivatal, NKFIH Funding text 1: The authors greatly thank Mária Vas (Institute of Enzymology, Research Center for Natural Sciences) for her critical review of the manuscript, Attila Bóta (Institute of Materials and Environmental Chemistry, Research Center for Natural Sciences) for his expert help in preliminary SAXS experiments. We also thank Nelly R. Hajizadeh and Dmitri I. Svergun at the EMBL SAXS facility for their professional help. The present work was supported by grants K108642, K128262, K134711, PD124451, and 2017-1.2.1-NKP-2017-00002 provided by National Research, Development and Innovation Office (NKFIH). AB - Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Its functions can be divided into two categories: membrane-proximal and membrane-distal. A recent study concluded that membrane-distal functions require the fully extended conformation, and this conclusion was supported by electron microscopy. The present solution small-angle X-ray scattering (SAXS) study revealed that ROCK2 population is a dynamic mixture of folded and partially extended conformers. Binding of RhoA to the coiled-coil domain shifts the equilibrium towards the partially extended state. Enzyme activity measurements suggest that the binding of natural protein substrates to the kinase domain breaks up the interaction between the N-terminal kinase and C-terminal regulatory domains, but smaller substrate analogues do not. The present study reveals the dynamic behaviour of this long, dimeric molecule in solution, and our structural model provides a mechanistic explanation for a set of membrane-proximal functions while allowing for the existence of an extended conformation in the case of membrane-distal functions. LA - English DB - MTMT ER - TY - JOUR AU - Somogyi, Márk AU - Szimler, Tamás AU - Baksa, Attila AU - Végh, Barbara AU - Bakos, Tamás AU - Paréj, Katalin AU - Ádám, Csaba AU - Zsigmond, Áron AU - Megyeri, Márton AU - Flachner, Beáta AU - Sajó, Ráchel AU - Gráczer, Éva Laura AU - Závodszky, Péter AU - Hajdú, István AU - Beinrohr, László TI - A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts JF - PLOS ONE J2 - PLOS ONE VL - 14 PY - 2019 IS - 12 PG - 17 SN - 1932-6203 DO - 10.1371/journal.pone.0227110 UR - https://m2.mtmt.hu/api/publication/31036023 ID - 31036023 LA - English DB - MTMT ER - TY - JOUR AU - Szimler, Tamás AU - Gráczer, Éva Laura AU - Györffy, Dániel AU - Végh, Barbara AU - Szilágyi, András AU - Hajdú, István AU - Závodszky, Péter AU - Kazinczyné Vas, Mária TI - New type of interaction between the SARAH domain of the tumour suppressor RASSF1A and its mitotic kinase Aurora A JF - SCIENTIFIC REPORTS J2 - SCI REP VL - 9 PY - 2019 PG - 12 SN - 2045-2322 DO - 10.1038/s41598-019-41972-x UR - https://m2.mtmt.hu/api/publication/30625253 ID - 30625253 LA - English DB - MTMT ER - TY - JOUR AU - Gráczer, Éva Laura AU - Szimler, Tamás AU - Garamszegi, A AU - Konarev, PV AU - Krezinger, Anikó AU - Oláh, Julianna AU - Pallo, A AU - Svergun, DI AU - Merli, A AU - Závodszky, Péter AU - Weiss, MS AU - Kazinczyné Vas, Mária TI - Dual Role of the Active Site Residues of Thermus thermophilus 3-Isopropylmalate Dehydrogenase: Chemical Catalysis and Domain Closure. JF - BIOCHEMISTRY J2 - BIOCHEMISTRY-US VL - 55 PY - 2016 IS - 3 SP - 560 EP - 574 PG - 15 SN - 0006-2960 DO - 10.1021/acs.biochem.5b00839 UR - https://m2.mtmt.hu/api/publication/3023735 ID - 3023735 AB - The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the kcat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (Km values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1-13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme-substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure. LA - English DB - MTMT ER - TY - JOUR AU - Gráczer, Éva Laura AU - Palló, Anna AU - Oláh, Julianna AU - Szimler, Tamás AU - Konarev, Petr V AU - Svergun, Dmitri I AU - Merli, Angelo AU - Závodszky, Péter AU - Weiss, Manfred S AU - Kazinczyné Vas, Mária TI - Glutamate 270 plays an essential role in K+-activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase JF - FEBS LETTERS J2 - FEBS LETT VL - 589 PY - 2015 IS - 2 SP - 240 EP - 245 PG - 6 SN - 0014-5793 DO - 10.1016/j.febslet.2014.12.005 UR - https://m2.mtmt.hu/api/publication/2807961 ID - 2807961 LA - English DB - MTMT ER - TY - JOUR AU - Palló, A AU - Oláh, Julianna AU - Gráczer, Éva Laura AU - Merli, A AU - Závodszky, Péter AU - Weiss, MS AU - Kazinczyné Vas, Mária TI - Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis JF - FEBS JOURNAL J2 - FEBS J VL - 281 PY - 2014 IS - 22 SP - 5063 EP - 5076 PG - 14 SN - 1742-464X DO - 10.1111/febs.13044 UR - https://m2.mtmt.hu/api/publication/2785761 ID - 2785761 N1 - Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Budapest, Hungary Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt. 2, Budapest, H-1117, Hungary Dipartimento di Bioscienze, Universitá Degli Studi di Parma, Parma, Italy Helmholtz Zentrum Berlin für Materialien und Energie, Macromolecular Crystallography, Albert Einstein Straße 15, Berlin, D-12489, Germany Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, B-1050, Belgium Brussels Center for Redox Biology, Brussels, B-1050, Belgium Structural Biology Brussels Laboratory, Vrije Universiteit Brussel, Brussels, B-1050, Belgium Cited By :12 Export Date: 15 April 2021 CODEN: FJEOA Correspondence Address: Weiss, M.S.; Helmholtz Zentrum Berlin für Materialien und Energie, Albert Einstein Straße 15, Germany; email: manfred.weiss@helmholtz-berlin.de Chemicals/CAS: 3 isopropylmalate dehydrogenase, 9030-97-1; magnesium, 7439-95-4; manganese, 16397-91-4, 7439-96-5; nicotinamide adenine dinucleotide, 53-84-9; potassium, 7440-09-7; 3-Isopropylmalate Dehydrogenase; Bacterial Proteins; Magnesium; Malates; Manganese; NAD; Potassium Funding details: Hungarian Scientific Research Fund, OTKA, 108642 AB - The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn2+, its substrate isopropylmalate and its co-factor product NADH at 2.0 Å resolution features a fully closed conformation of the enzyme. Upon closure of the two domains, the substrate and the co-factor are brought into precise relative orientation and close proximity, with a distance between the C2 atom of the substrate and the C4N atom of the pyridine ring of the co-factor of approximately 3.0 Å. The structure further shows binding of a K+ ion close to the active site, and provides an explanation for its known activating effect. Hence, this structure is an excellent mimic for the enzymatically competent complex. Using high-level QM/MM calculations, it may be demonstrated that, in the observed arrangement of the reactants, transfer of a hydride from the C2 atom of 3-isopropylmalate to the C4N atom of the pyridine ring of NAD+ is easily possible, with an activation energy of approximately 15 kcal·mol-1. The activation energy increases by approximately 4-6 kcal·mol-1 when the K+ ion is omitted from the calculations. In the most plausible scenario, prior to hydride transfer the ε-amino group of Lys185 acts as a general base in the reaction, aiding the deprotonation reaction of 3-isopropylmalate prior to hydride transfer by employing a low-barrier proton shuttle mechanism involving a water molecule. LA - English DB - MTMT ER - TY - JOUR AU - Gráczer, Éva Laura AU - Bacsó, András AU - Kónya, Dénes AU - Kazi, A AU - Soós, Tibor AU - Molnár, Laura AU - Szimler, Tamás AU - Beinrohr, László AU - Szilágyi, András AU - Závodszky, Péter AU - Kazinczyné Vas, Mária TI - Drugs Against Mycobacterium Tuberculosis 3-Isopropylmalate Dehydrogenase Can be Developed using Homologous Enzymes as Surrogate Targets JF - PROTEIN AND PEPTIDE LETTERS J2 - PROTEIN PEPTIDE LETT VL - 21 PY - 2014 IS - 12 SP - 1295 EP - 1307 PG - 13 SN - 0929-8665 UR - https://m2.mtmt.hu/api/publication/2604041 ID - 2604041 N1 - Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2, Budapest, H-1117, Hungary Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2, Budapest, H-1117, Hungary Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, P. O. Box 286, Budapest, H-1519, Hungary Cited By :2 Export Date: 3 October 2022 CODEN: PPELE Correspondence Address: Vas, M.; Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2, Hungary AB - 3-Isopropylmalate dehydrogenase (IPMDH) from Mycobacterium tuberculosis (Mtb) may be a target for specific drugs against this pathogenic bacterium. We have expressed and purified Mtb IPMDH and determined its physical-chemical and enzymological properties. Size-exclusion chromatography and dynamic light scattering measurements (DLS) suggest a tetrameric structure for Mtb IPMDH, in contrast to the dimeric structure of most IPMDHs. The kinetic properties (kcat and Km values) of Mtb IPMDH and the pH-dependence of kcat are very similar to both Escherichia coli (Ec) and Thermus thermophilus (Tt) IPMDHs. The stability of Mtb IPMDH in 8 M urea is close to that of the mesophilic counterpart, Ec IPMDH, both of them being much less stable than the thermophilic (Tt) enzyme. Two known IPMDH inhibitors, O-methyl oxalohydroxamate and 3-methylmercaptomalate, have been synthesised. Their inhibitory effects were found to be independent of the origin of IPMDHs. Thus, experiments with either Ec or Tt IPMDH would be equally relevant for designing specific inhibitory drugs against Mtb IPMDH. LA - English DB - MTMT ER - TY - JOUR AU - Gráczer, Éva Laura AU - Lionne, C AU - Zavodszky, P AU - Chaloin, L AU - Vas, M TI - Transient kinetics show isomerisation steps in the kinetic pathway of Isopropylmalate Dehydrogenase JF - EUROPEAN BIOPHYSICS JOURNAL J2 - EUR BIOPHYS J VL - 42 PY - 2013 IS - 1 SP - S176 EP - S176 SN - 0175-7571 UR - https://m2.mtmt.hu/api/publication/3027798 ID - 3027798 N1 - SU 1 LA - English DB - MTMT ER - TY - JOUR AU - Gráczer, Éva Laura AU - Lionne, Corinne AU - Závodszky, Péter AU - Chaloin, Laurent AU - Kazinczyné Vas, Mária TI - Transient kinetic studies reveal isomerization steps along the kinetic pathway of Thermusthermophilus 3-isopropylmalate dehydrogenase JF - FEBS JOURNAL J2 - FEBS J VL - 280 PY - 2013 IS - 8 SP - 1764 EP - 1772 PG - 9 SN - 1742-464X DO - 10.1111/febs.12191 UR - https://m2.mtmt.hu/api/publication/2339846 ID - 2339846 N1 - Megjegyzés-23169602 N1 : Chemicals/CAS3 isopropylmalate dehydrogenase, 9030-97-1; magnesium ion, 22537-22-0; reduced nicotinamide adenine dinucleotide, 58-68-4; tryptophan, 6912-86-3, 73-22-3 Megjegyzés-23169791 N1 : Chemicals/CAS3 isopropylmalate dehydrogenase, 9030-97-1; magnesium ion, 22537-22-0; reduced nicotinamide adenine dinucleotide, 58-68-4; tryptophan, 6912-86-3, 73-22-3 Megjegyzés-23174472 N1 : Chemicals/CAS3 isopropylmalate dehydrogenase, 9030-97-1; magnesium ion, 22537-22-0; reduced nicotinamide adenine dinucleotide, 58-68-4; tryptophan, 6912-86-3, 73-22-3 LA - English DB - MTMT ER - TY - JOUR AU - Matkovicsné Varga, Andrea AU - Gráczer, Éva Laura AU - Chaloin, L AU - Liliom, Károly AU - Závodszky, Péter AU - Lionne, C AU - Kazinczyné Vas, Mária TI - Selectivity of kinases on the activation of tenofovir, an anti-HIV agent JF - EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES J2 - EUR J PHARM SCI VL - 48 PY - 2013 IS - 1-2 SP - 307 EP - 315 PG - 9 SN - 0928-0987 DO - 10.1016/j.ejps.2012.11.007 UR - https://m2.mtmt.hu/api/publication/2226151 ID - 2226151 AB - Nucleoside analogues, used in HIV-therapy, need to be phosphorylated by cellular enzymes in order to become potential substrates for HIV reverse transcriptase. After incorporation into the viral DNA chain, because of lacking of their 3'- hydroxyl groups, they stop the elongation process and lead to the death of the virus. Phosphorylation of the HIV-drug derivative, tenofovir monophosphate was tested with the recombinant mammalian nucleoside diphosphate kinase (NDPK), 3- phosphoglycerate kinase (PGK), creatine kinase (CK) and pyruvate kinase (PK). Among them, only CK was found to phosphorylate tenofovir monophosphate with a reasonable rate (about 45-fold lower than with its natural substrate, ADP), while PK exhibits even lower, but still detectable activity (about 1000-fold lower compared to the value with ADP). On the other hand, neither NDPK nor PGK has any detectable activity on tenofovir monophosphate. The absence of activity with PGK is surprising, since the drug tenofovir competitively inhibits both CK and PGK towards their nucleotide substrates, with similar inhibitory constants, KI of 2.9 and 4.8 mM, respectively. Computer modelling (docking) of tenofovir mono- or diphosphate forms to these four kinases suggests that the requirement of large-scale domain closure for functioning (as for PGK) may largely restrict their applicability for phosphorylation/activation of pro-drugs having a structure similar to tenofovir monophosphate. © 2012 Elsevier B.V. All rights reserved. LA - English DB - MTMT ER -