UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

Autor: Leonardo Astolfi Rosado, Diana C. Rostirolla, Diógenes Santiago Santos, Ardala Breda, Luiz Augusto Basso, Mario Sergio Palma
Přispěvatelé: Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Universidade Estadual Paulista (Unesp)
Jazyk: angličtina
Rok vydání: 2011
Předmět:
molecular cloning
pyrimidine
gene amplification
Uridine Triphosphate
Guanosine triphosphate
Ligands
Biochemistry
Polymerase Chain Reaction
chemistry.chemical_compound
Adenosine Triphosphate
enzyme kinetics
protein purification
enzyme phosphorylation
Cloning
Molecular
Genes
Suppressor
Thermodynamic binding parameters
uridine 5' monophosphate kinase
Escherichia coli Proteins
Eukaryota
Allosteric regulation
Pyrimidine metabolism
unclassified drug
Cooperative kinetics
priority journal
Guanosine Triphosphate
Homotetramer
Spectrometry
Mass
Electrospray Ionization
adenosine triphosphate
Molecular Sequence Data
Biophysics
amino acid metabolism
gene sequence
Biology
Allosteric Regulation
Transferases
guanosine triphosphate
Escherichia coli
Enzyme kinetics
Amino Acid Sequence
phosphotransferase
Molecular Biology
protein expression
UMP kinase
uridine diphosphate
nonhuman
UMPK
Isothermal titration calorimetry
molecular weight
Mycobacterium tuberculosis
Sequence Analysis
DNA
Antitubercular drug target
Molecular Weight
Kinetics
Pyrimidines
chemistry
Adenosine triphosphate
Sequence Alignment
Zdroj: Scopus
Repositório Institucional da UNESP
Universidade Estadual Paulista (UNESP)
instacron:UNESP
Popis: Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:25:27Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:36:08Z : No. of bitstreams: 1 2-s2.0-78651254744.pdf: 1084067 bytes, checksum: 3e08679474341a3b002fd1821c21cc22 (MD5) Made available in DSpace on 2014-05-27T11:25:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-15 The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved. Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF) Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB) Pontifícia Universidade Católica Do Rio Grande Do sul (PUCRS), Av. Ipiranga 6681 - Tecnopuc - Prédio 92-A, Porto Alegre 90619-900, RS Programa de Pós-Graduaão em Biologia Celular e Molecular Pontifícia Universidade Católica Do Rio Grande Do sul (PUCRS), Porto Alegre, RS Programa de Pós-Graduaão em Medicina e Ciências da Saúde PUCRS, Av. Ipiranga 6681, Porto Alegre 90619-900, RS Laboratório de Biologia Estrutural e Zooquímica Centro de Estudos de Insetos Sociais Universidade Estadual Paulista (UNESP), Rio Claro, SP Laboratório de Biologia Estrutural e Zooquímica Centro de Estudos de Insetos Sociais Universidade Estadual Paulista (UNESP), Rio Claro, SP
Databáze: OpenAIRE
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