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41.
A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl-ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 10(3) and 10(5)-fold decreases in k(cat) observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30-fold decrease in activity and a 16-fold inflation of the K(m) for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphom-evalonate kinase (GHMP kinase) family. 相似文献
42.
Enterococcus faecalis mevalonate kinase 总被引:1,自引:0,他引:1
Gram-positive pathogens synthesize isopentenyl diphosphate, the five-carbon precursor of isoprenoids, via the mevalonate pathway. The enzymes of this pathway are essential for the survival of these organisms, and thus may represent possible targets for drug design. To extend our investigation of the mevalonate pathway in Enterococcus faecalis, we PCR-amplified and cloned into pET-28b the mvaK1 gene thought to encode mevalonate kinase, the fourth enzyme of the pathway. Following transformation of the construct EFK1-pET28b into Escherichia coli BL21(DE3) cells, the expressed C-terminally hexahistidine-tagged protein was purified on a nickel affinity support to apparent homogeneity. The purified protein catalyzed the divalent ion-dependent phosphorylation of mevalonate to mevalonate 5-phosphate. The specific activity of the purified kinase was 24 micromole/min/mg protein. Based on sedimentation velocity data, E. faecalis mevalonate kinase exists in solution primarily as a monomer with a mass of 32.2 kD. Optimal activity occurred at pH 10 and at 37 degrees C. Delta H(a) was 22 kcal/mole. Kinetic analysis suggested that the reaction proceeds via a sequential mechanism. K(m) values were 0.33 mM (mevalonate), 1.1 mM (ATP), and 3.3 mM (Mg(2+)). Unlike mammalian mevalonate kinases, E. faecalis mevalonate kinase utilized all tested nucleoside triphosphates as phosphoryl donors. ADP, but not AMP, inhibited the reaction with a K(i) of 2.7 mM. 相似文献
43.
The yeast farnesyl diphosphate synthase (FPPS) gene was engineered so as to construct allelic forms giving various activities of the enzyme. One of the substitutions was F96W in the chain length determination region. The other, K197, conserved within a consensus sequence found in the majority of FPP and GGPP synthases, was substituted by R, E and V. An intricate correlation has been found between the FPPS activity, the amount of ergosterol synthesized and cell growth of a mutant strain defective in FPPS. About 40% of wt FPPS activity was sufficient to support normal growth of the mutant. With further decline of FPPS activity (20 down to 3%) the amount of ergosterol remained unchanged at approximately 0.16% (vs dry weight), whereas growth yield decreased and lag times increased. We postulate that, in addition to ergosterol initiating and maintaining growth of yeast cells, FPP and/or its derivatives participate in these processes. 相似文献
44.
Phillips MA Wildung MR Williams DC Hyatt DC Croteau R 《Archives of biochemistry and biophysics》2003,411(2):267-276
The complex mixture of monoterpenes, sesquiterpenes, and diterpenes that comprises oleoresin provides the primary defense of conifers against bark beetles and their associated fungal pathogens. Monoterpene synthases produce the turpentine fraction of oleoresin, which allows mobilization of the diterpene resin acid component (rosin) and is also toxic toward invading insects; this is particularly the case for alpha-pinene, a prominent bicyclic monoterpene of pine turpentine. The stereochemistry of alpha-pinene is a critical determinant of host defense capability and has implications for host selection, insect pheromone biosynthesis, and tritrophic-level interactions. Pines produce both enantiomers of alpha-pinene, which appear to arise through antipodal reaction mechanisms by distinct enzymes. Using a cDNA library constructed with mRNA from flushing needles of loblolly pine (Pinus taeda), we employed a homology-based cloning strategy to isolate, and confirm by functional expression, the genes encoding (+)-(3R:5R)-alpha-pinene synthase, (-)-(3S:5S)-alpha-pinene synthase, and several other terpene synthases. The pinene synthases, which produce mirror-image products, share only 66% amino acid identity (72% similarity) but are similar in general properties to other monoterpene synthases of gymnosperms. The stereochemical control of monoterpene cyclization reactions, the evolution of "antipodal" enzymes, and the implications of turpentine composition in ecological interactions are discussed. 相似文献
45.
Kooistra O Bedoux G Brecker L Lindner B Sánchez Carballo P Haras D Zähringer U 《Carbohydrate research》2003,338(23):2667-2677
Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised. 相似文献
46.
Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease 总被引:4,自引:0,他引:4
Choroideremia is an X-chromosome-linked disease that leads to the degeneration of the choriocapillaris, the retinal pigment epithelium and the photoreceptor layer in the eye. The gene product defective in choroideremia, CHM, is identical to Rab escort protein 1 (REP1). CHM/REP1 is an essential component of the catalytic geranylgeranyltransferase II complex (GGTrII) that delivers newly synthesized small GTPases belonging to the RAB gene family to the catalytic complex for post-translational modification. CHM/REP family members are evolutionarily related to members of the guanine nucleotide dissociation inhibitor (GDI) family, proteins involved in the recycling of Rab proteins required for vesicular membrane trafficking through the exocytic and endocytic pathways, forming the GDI/CHM superfamily. Biochemical and structural analyses have now revealed a striking parallel in the organization and function of these two families allowing us to generate a general model for GDI/CHM superfamily function in health and disease. 相似文献
47.
Two recombinant sesquiterpene synthases from grand fir, delta-selinene synthase and gamma-humulene synthase, each produce more than 30 sesquiterpene olefins from the acyclic precursor farnesyl diphosphate. These enzymes contain a pair of DDxxD motifs, on opposite lips of the presumptive active site, which are thought to be involved in substrate binding and could promote multiple orientations of the substrate alkyl chain from which multiple families of cyclic olefins could derive. Mutagenesis of the first aspartate of either DDxxD motif resulted in depressed k(cat), with lesser effect on K(m), for delta-selinene synthase and afforded a much simpler product spectrum composed largely of monocyclic olefins. Identical alterations in gamma-humulene synthase produced similar kinetic effects with a simplified product spectrum of mostly acyclic and monocyclic olefins. Although impaired in product diversity, none of the mutant synthases lost entirely the capacity to generate complex structures. These results confirm the catalytic significance of the DDxxD motifs and imply that they also influence permitted modes of cyclization. Deletion of an N-terminal arginine pair in delta-selinene synthase (an element potentially involved in substrate isomerization) altered kinetics without substantially altering product outcome. Finally, mutation of an active-site tyrosine residue thought to play a role in proton exchange had little influence; however, substitution of a nearby active site aspartate dramatically altered kinetics and product outcome. 相似文献
48.
Human brain nucleoside diphosphate kinase activity is decreased in Alzheimer's disease and Down syndrome 总被引:4,自引:0,他引:4
Kim SH Fountoulakis M Cairns NJ Lubec G 《Biochemical and biophysical research communications》2002,296(4):970-975
In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms. 相似文献
49.
Ying Shan Han Robert van der Heijden Alfons W M Lefeber Cornelis Erkelens Robert Verpoorte 《Phytochemistry》2002,59(1):45-55
Robustaquinone B was found as a major anthraquinone in cell cultures of Cinchona 'Robusta' after treatment with a fungal elicitor. Anthraquinones in Cinchona are considered to be of the Rubia type, i.e. rings A and B are derived from chorismate and alpha-ketoglutarate, whereas ring C is formed from isopentenyl diphosphate (IPP). To determine the origin of IPP, either formed via the mevalonic acid pathway or the 2-C-methyl-D-erythritol 4-phosphate pathway, the incorporation of [1-13C]glucose into robustaquinone B was studied. The 13C labeling of robustaquinone B was analyzed by one- and two-dimensional NMR spectroscopy and the labeling pattern was compared with the hypothetical labeling patterns obtained via the different biosynthetic pathways. The results clearly show that the IPP, constituting the ring C of robustaquinone B, is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. Moreover, the data also confirm that rings A and B of robustaquinone B are formed from chorismate and alpha-ketoglutarate via o-succinylbenzoate. 相似文献
50.