Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ferredoxin-NADP+ reductase from the cyanobacterium Anabaena sp. PCC 7119 was chemically modified by the alpha-dicarbonyl reagent phenylglyoxal. The studies of the inactivation by this compound, which is specific for arginyl residues, of both the diaphorase and NADPH-cytochrome c reductase activities, characteristic of the enzyme, are indicative of the involvement of at least one group of this kind in the binding site of NADP+ and a second one implicated in the interaction with ferredoxin. After specific cleavage of a FNR sample incubated with [7-14C]phenylglyoxal, two major labeled peptides were identified. The peptide which exhibited the higher degree of modification corresponded to residues 208-242. It contained four arginine residues but only two of them were the target of the modification: Arg224 and Arg233. Protection studies with protein substrates and sequence comparison with other reductases allow us to propose that these residues in Anabaena sp. PCC 7119 FNR must be involved in the interaction with the pyridine nucleotide. The second peptide corresponds to residues 75-103 and although it contains three arginine residues, Arg77 is the only one that exhibits the modification. This residue seems to be a key one in the interaction of this reductase with ferredoxin.
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PMID:Identification of arginyl residues involved in the binding of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 to its substrates. 144 67

Ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 is chemically modified by pyridoxal 5'-phosphate. The incorporation of 2 +/- 0.3 mol pyridoxal 5'-phosphate/mol ferredoxin-NADP+ reductase inhibited NADPH-cytochrome c reductase activity by up to 95% while 55% of diaphorase activity still remained. Considerable protection against inactivation was afforded by ferredoxin. Chymotryptic cleavage of the modified enzyme was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their fluorescence and by their absorbance at 325 nm. Three major labelled peptides were found. Their sequences were comprised of residues 46-54, 231-235 and 289-295. Lys-53 and -294 were the residues which presented the highest degree of modification and seem to be involved in the ferredoxin binding site of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119.
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PMID:Lysine residues on ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding. 154 17

The ndhC and ORF159 genes of the maize plastid DNA (ptDNA) were sequenced and maize ORF159 was used to screen a library of genomic DNA of the blue-green alga Synechocystis sp. PCC 6803. The cyanobacterial gene homologous to ORF159 (ORF157) was isolated and sequenced. In sequencing the region upstream of ORF157, reading frames with homology to the ndhC and psbG genes of maize ptDNA were identified. The ndhC and psbG genes overlap in the ptDNAs of maize, tobacco and Marchantia polymorpha, but are separated by a noncoding spacer in Synechocystis. Northern blot analysis showed that the ndhC, psbG and ORF157/159 genes are cotranscribed in maize and Synechocystis. The three genes occur in the same order in ptDNA of maize, tobacco, and M. polymorpha as in Synechocystis 6803. The amino acid sequences of the NDH-C, PSII-G and the ORF157/159 proteins deduced from the maize genes are 65%, 52% and 53% homologous to those of Synechocystis. However, the cyanobacterial and higher plant NDH-C protein sequences are only 23% homologous to the mitochondrial NDH-3 protein. Protein products of in vitro transcription/translation of the Synechocystis transcription unit had apparent molecular masses of 6 kDa (NDH-C), 25 kDa (PSII-G) and 22 kDa (ORF157) on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis. If these are components of an NADH dehydrogenase, cyanobacteria appear to resemble mitochondria more than they do Escherichia coli and Rhodopseudomonas capsulata with regard to this enzyme complex.
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PMID:Characterization of the ndhC-psbG-ORF157/159 operon of maize plastid DNA and of the cyanobacterium Synechocystis sp. PCC6803. 249 64

A part of the tRNA(Leu)(UAA) gene containing a 240-nucleotide group I intron was amplified by PCR from cyanobacterium Synechococcus PCC 6301 genomic DNA. The pre-tRNA synthesized from the cloned PCR product was efficiently self-spliced in vitro under physiological conditions. The gene encoding the tRNA(Leu)(UAA), trnL-UAA, was isolated from a Synechococcus PCC 6301 genomic library and the nucleotide sequence of a 2,167-bp portion was determined. The trnL-UAA consists of a 34-bp 5' exon, a 240-bp group I intron and a 50-bp 3' exon. In addition, three open reading frames (ORF1, ORF2 and ORF3) were found in the 5' and 3' flanking regions of trnL-UAA. The predicted protein sequence of ORF3, which is located 74-bp upstream from trnL-UAA on the opposite strand, shows 66.2% amino acid identity to that of the Synechocystis PCC 6803 gene encoding subunit L of NADH dehydrogenase (ndhL).
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PMID:Genes encoding the group I intron-containing tRNA(Leu) and subunit L of NADH dehydrogenase from the cyanobacterium Synechococcus PCC 6301. 758 50

The high-concentration CO2-requiring mutant N5 of Synechococcus sp. PCC 7942 was obtained by the insertion of a kanamycin-resistant gene at the EcoRI site, 12.4 kb upstream of rbc. The mutant is unable to accumulate inorganic carbon internally and exhibits very low apparent photosynthetic affinity for inorganic carbon but a photosynthetic Vmax similar to that of the wild type. Sequence and northern analyses showed that the insertion inactivated a gene highly homologous to ndhB, encoding subunit II of NADH dehydrogenase in Synechocystis sp. PCC 6803 (T. Ogawa [1991] Proc Natl Acad Sci USA 88: 4275-4279). When the mutant and the wild-type cells were exposed to 5% CO2 in air, their photosynthetic electron transfer capabilities, as revealed by fluorescence and thermoluminescence measurements, were similar. On the other hand, a significant decrease in variable fluorescence was observed when the mutant (but not the wild-type) cells were exposed to low CO2 under continuous light. The same treatment also resulted in a shift (from 38-27 degrees C) in the temperature at which the maximal thermoluminescence emission signal was obtained in the mutant but not in the wild type. These results may indicate that subunit II of NADH dehydrogenase is essential for the functional operation of the photosynthetic electron transport in Synechococcus under low but not high levels of CO2. We suggest that the inability to accumulate inorganic carbon under air conditions stems from disrupture of electron transport in this mutant.
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PMID:High CO2 concentration alleviates the block in photosynthetic electron transport in an ndhB-inactivated mutant of Synechococcus sp. PCC 7942. 831 46

The ndhF gene of the unicellular marine cyanobacterium Synechococcus sp. strain PCC 7002 was cloned and characterized. NdhF is a subunit of the type 1, multisubunit NADH:plastoquinone oxidoreductase (NADH dehydrogenase). The nucleotide sequence of the gene predicts an extremely hydrophobic protein of 664 amino acids with a calculated mass of 72.9 kDa. The ndhF gene was shown to be single copy and transcribed into a monocistronic mRNA of 2,300 nucleotides. An ndhF null mutation was successfully constructed by interposon mutagenesis, demonstrating that NdhF is not required for cell viability under photoautotrophic growth conditions. The mutant strain exhibited a negligible rate of oxygen uptake in the dark, but its photosynthetic properties (oxygen evolution, chlorophyll/P700 ratio, and chlorophyll/P680 ratio) were generally similar to those of the wild type. Although the ndhF mutant strain grew as rapidly as the wild-type strain at high light intensity, the mutant grew more slowly than the wild type at lower light intensities and did not grow at all under photoheterotrophic conditions. The roles of the NADH:plastoquinone oxidoreductase in photosynthetic and respiratory electron transport are discussed.
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PMID:Isolation and characterization of the ndhF gene of Synechococcus sp. strain PCC 7002 and initial characterization of an interposon mutant. 850 Oct 38

The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E. coli. Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies. The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR. Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin. Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR. A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation. In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor.
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PMID:Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis. 951 8

The bidirectional, NAD+-dependent hydrogenase from cyanobacteria is encoded by the structural genes hoxFUYH, which have been found to be clustered, though interspersed with different open reading frames (ORFs), in the heterocystous, N2-fixing Anabaena variabilis and in the unicellular Synechocystis PCC 6803. In another unicellular, non N2-fixing cyanobacterium, Anacystis nidulans, hoxF has now been identified as being separated by at least 16 kb from the residual structural genes hoxUYH. An ORF (termed hoxE gene) is located immediately upstream of hoxF in A. nidulans and in Synechocystis. Its deduced amino acid sequence shows similarities to the NuoE subunit of NADH dehydrogenase I of E. coli, to the homologous subunit of respiratory complex I in mitochondria, and also to the first 104 amino acids of HoxF in A. nidulans and Synechocystis. The diversity in the arrangement of hydrogenase genes in cyanobacteria is puzzling. The subunits HoxE, HoxF, and HoxU of the diaphorase part of the bidirectional hydrogenase have been discussed to be shared both by respiratory complex I and bidirectional hydrogenase in cyanobacteria. Different hoxU mutants were obtained by inserting a lacZKmR cassette into the gene both in A. nidulans and Anacystis PCC 7942. Such mutants showed reduced H2-evolution activities catalyzed by the bidirectional hydrogenase, but had nonimpaired respiratory O2-uptake. A common link between respiratory complex I and the diaphorase part of the bidirectional hydrogenase in cyanobacteria may still exist, but this hypothesis could not be verified in the present study by analyzing defined mutants impaired in one of the diaphorase genes.
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PMID:Unusual gene arrangement of the bidirectional hydrogenase and functional analysis of its diaphorase subunit HoxU in respiration of the unicellular cyanobacterium anacystis nidulans 954 59

Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR), indicate that the positive charge of Lys75 might be directly involved in the interaction between FNR and its protein partners, ferredoxin (Fd) and flavodoxin (Fld). To assess this possibility, this residue has been replaced by another positively charged residue, Arg, by two uncharged residues, Gln and Ser, and by a negatively charged residue, Glu. UV-vis absorption, fluorescence, and CD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Lys75Ser, and Lys75Glu) indicate that all the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Steady-state kinetic parameters for these FNR mutants, utilizing the diaphorase activity with DCPIP, indicate that Lys75 is not a critical residue for complex formation and electron transfer (ET) between FNR and NADP+ or NADPH. However, steady-state kinetic activities requiring complex formation and ET between FNR and Fd or Fld were appreciably affected when the positive charge at position of Lys75 was removed, and the ET reaction was not even measurable if a negatively charged residue was placed at this position. These kinetic parameters also suggest that it is complex formation that is affected by mutation. Consistent with this, when dissociation constants (Kd) for FNRox-Fdox (differential spectroscopy) and FNRox-Fdrd (laser flash photolysis) were measured, it was found that neutralization of the positive charge at position 75 increased the Kd values by 50-100-fold, and that no complex formation could be detected upon introduction of a negative charge at this position. Fast transient kinetic studies also corroborated the fact that removal of the positive charge at position 75 of FNR appreciably affects the complex formation process with its protein partners but indicates that ET is still achieved in all the reactions. This study thus clearly establishes the requirement of a positive charge at position Lys75 for complex formation during ET between FNR and its physiological protein partners. The results also suggest that the interaction of this residue with its protein partners is not structurally specific, since Lys75 can still be efficiently substituted by an arginine, but is definitely charge specific.
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PMID:Lys75 of Anabaena ferredoxin-NADP+ reductase is a critical residue for binding ferredoxin and flavodoxin during electron transfer. 975 47

The Synechocystis sp. PCC 6803 triple mutant D2R8 with V247M/A249T/M329I mutations in the D2 subunit of the photosystem II is impaired in Q(A) function, has an apparently mobile Q(A), and is unable to grow photoautotrophically. Several photoautotrophic pseudorevertants of this mutant have been isolated, each of which retained the original psbDI mutations of D2R8. Using a newly developed mapping technique, the site of the secondary mutations has been located in the open reading frame slr0399. Two different nucleotide substitutions and a deletion of about 60% of slr0399 were each shown to restore photoautotrophy in different pseudorevertants of the mutant D2R8, suggesting that inactivation of Slr0399 leads to photoautotrophic growth in D2R8. Indeed, a targeted deletion of slr0399 restores photoautotrophy in D2R8 and in other psbDI mutants impaired in Q(A) function. Slr0399 is similar to the hypothetical protein Ycf39, which is encoded in the cyanelle genome of Cyanophora paradoxa; in the chloroplast genomes of diatoms, dinoflagellates, and red algae; and in the nuclear genome of Arabidopsis thaliana. Slr0399 and Ycf39 have a NAD(P)H binding motif near their N terminus and have some similarity to isoflavone reductase-like proteins and to a subunit of the eukaryotic NADH dehydrogenase complex I. Deletion of slr0399 in wild type Synechocystis sp. PCC 6803 has no significant phenotypic effects other than a decrease in thermotolerance under both photoautotrophic and photomixotrophic conditions. We suggest that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around Q(A) in photosystem II. Moreover, as the effects of Slr0399 are not limited to photosystem II, this protein may also be involved in assembly of quinones in other photosynthetic and respiratory complexes.
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PMID:Inactivation of the open reading frame slr0399 in Synechocystis sp. PCC 6803 functionally complements mutations near the Q(A) niche of photosystem II. A possible role of Slr0399 as a chaperone for quinone binding. 1052 36


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