Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This communication describes the isolation and characterization of mutants of Rhizobium trifolii which can induce nitrogenase activity in defined liquid medium. Two procedures were used for the isolation of these mutants from R. trifolii strain DT-6: (1) following chemical mutagenesis, slow growing mutants were selected which were unable to utilize NH+4 as sole source of nitrogen; (2) as spontaneous mutants resistant to the glutamate analogue L-methionine-DL-sulfoximine. Mutants (DT-71, DT-125) isolated by these procedures induced nitrogenase activity in the free-living state, whereas the parent strain lacked this property. Induction of nitrogenase activity in these mutants occurred during the late exponential phase of growth when the rate of protein synthesis was decreasing. The addition of NH+4 to a medium containing glutamate as the nitrogen-source resulted in a 50--70% reduction (repression?) of nitrogenase activity; in contrast, the rate of protein synthesis or the rate of respiration was not influenced by exogenous NH+4. Biochemical analysis showed that these mutants (strains DT-71 and DT-125) have defects in both nitrogen and carbon metabolism. The levels of glutamate synthase (both NADP+ -and NAD+ -dependent activities) and glutamate dehydrogenase (NAD+-dependent activity) were markedly lower. In addition, the mutants were found to have no detectable ribitol dehydrogenase or beta-galactosidase activity. These findings are discussed in relation to a mechanism of regulation of symbiotic nitrogen fixation.
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PMID:Regulation of nitrogen fixation in Rhizobium spp. Isolation of mutants of Rhizobium trifolii which induce nitrogenase activity. 58 92

A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.
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PMID:Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants. 162 2

The flavoprotein ferredoxin-NADP+ reductase (FNR) catalyzes the final step of the photosynthetic electron transport chain, i.e. the reduction of NADP+ by ferredoxin. A cloned FNR cDNA from a pea library (Newman, B., and Gray, J. (1988) Plant Mol. Biol. 10, 511-520) was used to construct plasmids which express the apoenzyme in Escherichia coli. Two recombinant vectors were prepared, one containing the sequence corresponding to the mature enzyme and another including, in addition, the sequence of the transit peptide that directs FNR to the chloroplast. These proteins were expressed as fusion products to the NH2-terminal portion of beta-galactosidase. In both cases, a 35-kDa immunoreactive polypeptide was the major product, suggesting that the proteins were processed in vivo. NH2-terminal sequence determination of the purified recombinant proteins indicate cleavage at positions -1/-2 with respect to the normal processing site in chloroplasts. The processed enzymes showed enzymatic activities and spectral properties that were similar or identical to those of native plant FNR. When a La protease-deficient E. coli strain was used as a host, the expressed FNR precursor was found to be poorly processed, associated to bacterial pellets, and showed no detectable FNR activity. The overall results indicate that acquisition of the native enzyme conformation and assembly of the prosthetic group takes place in the bacterial host, generating an enzyme that is, as far as studied, indistinguishable from plant FNR.
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PMID:Expression, assembly, and processing of an active plant ferredoxin-NADP+ oxidoreductase and its precursor protein in Escherichia coli. 190 76

Sepiapterin reductase (7,8-dihydrobiopterin: NADP+ oxidoreductase, EC 1.1.1.153) catalyzes the terminal step in the biosynthetic pathway for tetrahydrobiopterin, the cofactor necessary for aromatic amino acid hydroxylation. We report here the isolation of a cDNA clone for rat liver sepiapterin reductase. The cDNA has been excised from a lambda vector and the DNA sequence was determined. The insert contains the coding sequence for at least 95% of the rat enzyme and is fused to the Escherichia coli beta-galactosidase N-terminal segment and the lac promoter. The N-terminal region of the clone contains an extraordinarily high G + C content. The amino acid sequence deduced from the clone is in agreement with the size and composition of the enzyme and was matched to several tryptic peptide sequences. The enzyme encoded by the cDNA insert was shown to have sepiapterin reductase activity after expression in E. coli. Structural similarities were identified between this protein and several enzymes that should contain similar nucleotide and pteridine binding sites.
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PMID:Isolation and expression of rat liver sepiapterin reductase cDNA. 220 Oct 30

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.
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PMID:Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase. 1633 84