RESUMO
Human cytosolic NADP(+)-dependent malic enzyme (c-NADP-ME) is neither a cooperative nor an allosteric enzyme, whereas mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by fumarate. This study examines the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME and c-NADP-ME. Multiple residues corresponding to the fumarate-binding site were mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME. Additionally, the crystal structure of the apo (ligand-free) human c-NADP-ME conformation was determined. Kinetic studies indicated no significant difference between the wild-type and mutant enzymes in Km,NADP, Km,malate, and kcat. A chimeric enzyme, [51-105]_c-NADP-ME, was designed to include the putative fumarate-binding site of m-NAD(P)-ME at the dimer interface of c-NADP-ME; however, this chimera remained nonallosteric. In addition to fumarate activation, the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME is quite different; c-NADP-ME is a stable tetramer, whereas m-NAD(P)-ME exists in equilibrium between a dimer and a tetramer. The quaternary structures for the S57K/N59E/E73K/S102D and S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G mutants of c-NADP-ME are tetrameric, whereas the K57S/E59N/K73E/D102S m-NAD(P)-ME quadruple mutant is primarily monomeric with some dimer formation. These results strongly suggest that the structural features near the fumarate-binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME. In this study, we attempt to delineate the structural features governing the fumarate-induced allosteric activation of malic enzyme.
RESUMO
Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P)-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P)-ME_R67A/R91A and m-NAD(P)-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P)-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P)-ME enzyme activity.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fumaratos/química , Fumaratos/farmacologia , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/química , Mitocôndrias/enzimologia , Regulação Alostérica/efeitos dos fármacos , Citosol/enzimologia , HumanosRESUMO
Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated horses. Therefore, the EIV H3N8 subtype specific iiRT-PCR assay along with the portable POCKIT™ Nucleic Acid Analyzer provides a highly reliable, sensitive and specific on-site detection system of both equine and canine influenza viruses.