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91.
Ketopantoate reductase (EC 1.1.1.169), an enzyme in the pantothenate biosynthetic pathway, catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate. The enzyme exhibits high specificity for ketopantoate, with V and V/K for ketopantoate being 5- and 365-fold higher than those values for alpha-ketoisovalerate and 20- and 648-fold higher than those values for alpha-keto-beta-methyl-n-valerate, respectively. For pyridine nucleotides, V/K for beta-NADPH is 3-500-fold higher than that for other nucleotide substrates. The magnitude of the primary deuterium kinetic isotope effects on V and V/K varied substantially when different ketoacid and pyridine nucleotide substrates were used. The small primary deuterium kinetic isotope effects observed using NADPH and NHDPH suggest that the chemical step is not rate-limiting, while larger primary deuterium isotope effects were observed for poor ketoacid and pyridine nucleotide substrates, indicating that the chemical reaction has become partially or completely rate-limiting. The pH dependence of (D)V using ketopantoate was observed to vary from a value of 1.1 at low pH to a value of 2.5 at high pH, while the magnitude of (D)V/K(NADPH) and (D)V/K(KP) were pH-independent. The value of (D)V is large and pH-independent when alpha-keto-beta-methyl-n-valerate was used as the ketoacid substrate. Solvent kinetic isotope effects of 2.2 and 1.2 on V and V/K, respectively, were observed with alpha-keto-beta-methyl-n-valerate. Rapid reaction analysis of NADPH oxidation using ketopantoate showed no "burst" phase, suggesting that product-release steps are not rate-limiting and the cause of the small observed kinetic isotope effects with this substrate pair. Large primary deuterium isotope effects on V and V/K using 3-APADPH in steady-state experiments, equivalent to the isotope effect observed in single turnover studies, suggests that chemistry is rate-limiting for this poorer reductant. These results are discussed in terms of a kinetic and chemical mechanism for the enzyme. 相似文献
92.
Dihydrodipicolinate reductase (DHPR) catalyzes the reduced pyridine nucleotide-dependent reduction of the alpha,beta-unsaturated cyclic imine, dihydrodipicolinate, to generate tetrahydrodipicolinate. This enzyme catalyzes the second step in the bacterial biosynthetic pathway that generates meso-diaminopimelate, a component of bacterial cell walls, and the amino acid L-lysine. The Mycobacterium tuberculosis dapB-encoded DHPR has been cloned, expressed, purified, and crystallized in two ternary complexes with NADH or NADPH and the inhibitor 2,6-pyridinedicarboxylate (2,6-PDC). The structures have been solved using molecular replacement strategies, and the DHPR-NADH-2,6-PDC and DHPR-NADPH-2,6-PDC complexes have been refined against data to 2.3 and 2.5 A, respectively. The M. tuberculosis DHPR is a tetramer of identical subunits, with each subunit composed of two domains connected by two flexible hinge regions. The N-terminal domain binds pyridine nucleotide, while the C-terminal domain is involved in both tetramer formation and substrate/inhibitor binding. The M. tuberculosis DHPR uses NADH and NADPH with nearly equal efficiency based on V/K values. To probe the nature of this substrate specificity, we have generated two mutants, K9A and K11A, residues that are close to the 2'-phosphate of NADPH. These two mutants exhibit decreased specificity for NADPH by factors of 6- and 30-fold, respectively, but the K11A mutant exhibits 270% of WT activity using NADH. The highly conserved structure of the nucleotide fold may permit other enzyme's nucleotide specificity to be altered using similar mutagenic strategies. 相似文献
93.
94.
Inducible nitric oxide synthase attenuates chronic colitis in human histocompatibility antigen HLA-B27/human beta2 microglobulin transgenic rats 总被引:1,自引:0,他引:1
95.
Beaman TW Vogel KW Drueckhammer DG Blanchard JS Roderick SL 《Protein science : a publication of the Protein Society》2002,11(4):974-979
Tetrahydrodipicolinate N-succinyltransferase (DapD) catalyzes the succinyl-CoA-dependent acylation of L-2-amino-6-oxopimelate to 2-N-succinyl-6-oxopimelate as part of the succinylase branch of the meso-diaminopimelate/lysine biosynthetic pathway of bacteria, blue-green algae, and plants. This pathway provides meso-diaminopimelate as a building block for cell wall peptidoglycan in most bacteria, and is regarded as a target pathway for antibacterial agents. We have solved the X-ray crystal structures of DapD in ternary complexes with pimelate/succinyl-CoA and L-2-aminopimelate with the nonreactive cofactor analog, succinamide-CoA. These structures define the binding conformation of the cofactor succinyl group and its interactions with the enzyme and place its thioester carbonyl carbon in close proximity to the nucleophilic 2-amino group of the acceptor, in support of a direct attack ternary complex mechanism. The acyl group specificity differences between homologous tetrahydrodipicolinate N-acetyl- and N-succinyltransferases can be rationalized with reference to at least three amino acids that interact with or give accessible active site volume to the cofactor succinyl group. These residues account at least in part for the substrate specificity that commits metabolic intermediates to either the succinylase or acetylase branches of the meso-diaminopimelate/lysine biosynthetic pathway. 相似文献
96.
Buckner CM Gettie A Tan RC Eshetu T Ratterree M Blanchard J Cheng-Mayer C Harouse JM 《Journal of medical primatology》2002,31(4-5):164-170
Infection of rhesus macaques with chimeric simian-human immunodeficiency viruses (SHIV) is an established model to study acquired immunodeficiency syndrome (AIDS) pathogenesis. Such a controlled system allows for detailed analysis of the molecular determinants of viral pathogenesis in addition to studying host-specific immune responses that modulate disease progression. Furthermore, the use of a pathogenic molecular clone affords the opportunity to study both viral evolution within a host and to examine the generation of tissue specific variants. In this report we describe viral diversification within tissues of two rhesus macaques infected intravenously with the CXCR4-specific molecular clone SHIVSF33A2. Heteroduplex tracking analysis (HTA) was used to determine the complexity of viral DNA within distinct lymphoid tissues. Not surprising, heterogeneity of the proviral quasispecies in tissues obtained during the acute infection was limited. However, tissues obtained at necropsy harbored a more diverse and often different population of env variants. As the inoculating virus is a molecular clone, the variants generated are likely due to the presence of tissue specific selective forces rather than a founder's effect. 相似文献
97.
The effects of a single or double regimen of exogenous progesterone and estradiol-17beta (P/E, total dose 300 mg P/20 mg E) were investigated in 50 postparturient Quarter Horse mares. In Trial 1, at 1 and 24 h after foaling, mares were injected with progesterone (150 mg) and estradiol-17beta (10 mg) (n = 7) or 0.9% NaCl (control, n = 13). In Trial 2, within 12 h after foaling, mares were injected with progesterone (300 mg) and estradiol-17beta (20 mg) (n = 13) or 0.9% NaCl (control, n = 17). Mares were examined daily by palpation per rectum and transrectal ultrasonography to determine the day of ovulation. The largest cross sectional diameters of each uterine horn and uterine body were measured ultrasonographically on Day 15 postpartum. Mean uterine diameters did not differ between treatment groups (P > 0.05) in Trial 1, Trial 2 or for combined data for both Trials 1 and 2. For mares bred on the first postpartum estrus pregnancy rates did not differ (P > 0.05) between treatment groups (16/18, 89%) and controls (22/30, 81%) nor was there a difference in mean day to first postpartum ovulation (P > 0.05) between treated and control groups in Trial 1, Trial 2 or Trials 1 and 2 combined. However, fewer (P < 0.05) total P/E treated mares (0/20) ovulated prior to Day 10 postpartum than did control mares (6/30). Variance in days to ovulation was lower (P < 0.05) for P/E treated mares (var = 3.73 days) than for control mares (var = 7.64 days) for data combined from Trials 1 and 2. 相似文献
98.
The immunological synapse: the more you look the less you know.. 总被引:3,自引:0,他引:3
Blanchard N Hivroz C 《Biology of the cell / under the auspices of the European Cell Biology Organization》2002,94(6):345-354
Before T cells of the immune system can recognize pathogens, antigen presenting cells (APCs) must process pathogen-derived peptides and present them together with major histocompatibility complex molecules (MHC) to T lymphocytes. T lymphocytes then scan the surface of APCs and antigen-specific activation of the T cell will happen after interaction of T cell antigen receptor (TCR) with MHC-peptide complexes expressed at the membrane of APCs. This interaction takes place in a nanometer scale gap between the two cells, referred to as an immunological synapse. Recent three-dimensional fluorescence analysis of this synapse revealed a dynamic spatial organization of membrane receptors, cytoskeleton and intracellular signaling complexes on the T cell side showing specific patterns, which depend on the nature of the T cell:APC pair. Although it is obvious that establishment of an intimate contact between T cells and APCs will facilitate cell:cell communication it is not clear what is the role, if any, of this receptors patterning. This molecular reorganization has long been thought to enhance and/or sustain TCR signaling and thus T cell activation, but this is now a matter of controversy. Moreover, mechanisms controlling immunological synapse formation are still unraveled. Segregation of proteins may occur spontaneously as proposed by mathematical modeling taking into account membrane fluidity, protein size and receptor/ligand affinity. Alternatively patterning of the molecules at the cell:cell interface could be driven by active processes involving T cell signaling and/or specific features of the APC. These different questions will be discussed herein. 相似文献
99.
Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle 总被引:14,自引:0,他引:14
Boëda B El-Amraoui A Bahloul A Goodyear R Daviet L Blanchard S Perfettini I Fath KR Shorte S Reiners J Houdusse A Legrain P Wolfrum U Richardson G Petit C 《The EMBO journal》2002,21(24):6689-6699
Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia. 相似文献
100.
Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions. 相似文献