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M F Fillat

Publications and source records attributed to M F Fillat.

At least 19 recordsLinked to original sources
PubMed2007

Expression of furA is modulated by NtcA and strongly enhanced in heterocysts of Anabaena sp. PCC 7120.

Fur (ferric uptake regulator) proteins are principally responsible for maintaining iron homeostasis in prokaryotes. Iron is usually a scarce resource. Its limitation reduces photosynthetic rates and cell growth in cyanobacteria in general and especially in cyanobacteria that are fixing dinitrogen, a process that requires the synthesis of numerous proteins with a high content of iron. This paper shows that in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120, levels of furA mRNA and FurA protein increase significantly in response to nitrogen deprivation, and that furA up-regulation takes place specifically in proheterocysts and mature heterocysts. Great differences in a Northern blot, probed with furA, of RNA from an ntcA mutant relative to wild-type Anabaena sp. were attributable to binding of NtcA, a global regulator of nitrogen metabolism, to the promoter of furA and to the promoter of the furA antisense transcript alr1690-alpha-furA.

Anabaena↗
PubMed2006

Interaction of FurA from Anabaena sp. PCC 7120 with DNA: a reducing environment and the presence of Mn(2+) are positive effectors in the binding to isiB and furA promoters.

The Fur (ferric uptake regulator) protein is a global regulator in most prokaryotes that controls a large number of genes. Fur is a classical repressor that uses ferrous iron as co-repressor and binds to specific DNA sequences (iron boxes) as a dimer. Three different genes coding for Fur homologues have been identified in Anabaena sp. PCC 7120. FurA controls the transcription of flavodoxin, the product of the isiB gene, and is moderately autoregulated. In this work, the promoter of the furA gene was defined and the FurA protected regions in the furA and isiB promoters were identified, showing that the binding sites for Anabaena FurA contain A/T-rich sequences with a variable arrangement compared to the conventional 19-base pair Fur consensus. The influence of different factors on the interaction between FurA and the promoters was evaluated in vitro. The affinity of FurA for the DNA targets was significantly affected by the redox status of this regulator and the presence of Mn(2+). The optimal binding conditions were observed in the presence of both Mn(2+) and DTT. Those results suggest that, in addition to iron availability, FurA-DNA interaction is modulated by redox conditions.

Anabaena↗
PubMed2001

Simultaneous occurrence of two different glyceraldehyde-3-phosphate dehydrogenases in heterocystous N(2)-fixing cyanobacteria.

Enzyme activity determinations and Western and Northern blot analyses have shown the presence of two catalytically different glyceraldehyde-3-phosphate dehydrogenases (GAPDH) in both vegetative cells and heterocysts of several N(2)-fixing Anabaena strains: (a) the gap2-encoded NAD(P)-dependent GAPDH2 (EC 1.2.1.59), the enzyme involved in the photosynthetic carbon assimilation pathway, which is present at higher levels in vegetative cells, and (b) the gap3-encoded NAD-dependent GAPDH3 (EC 1.2.1.12), presumably involved in carbohydrate anabolism and catabolism, which is the predominant GAPDH in heterocysts. In contrast, the gap1-encoded GAPDH1, which is the other NAD-dependent cyanobacterial GAPDH, is virtually absent in both cell types. These findings are discussed in the context of carbon metabolism of heterocystous N(2)-fixing cyanobacteria.

Anabaena↗
PubMed2001

Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter.

A gene coding for a Fur (ferric uptake regulation) protein from the cyanobacterium Anabaena PCC 7119 has been cloned and overexpressed in Escherichia coli. DNA sequence analysis confirmed the presence of a 151-amino-acid open reading frame that showed homology with the Fur proteins reported for the unicellular cyanobacteria Synechococcus 7942 and Synechocystis PCC 6803. Two putative Fur-binding sites were detected in the promoter regions of the fur gene from Anabaena. Partially purified recombinant Fur binds to the flavodoxin promoter as well as its own promoter. This suggests that the Fur gene is autoregulated in Anabaena.

Amino Acid Sequence↗
PubMed1999

Constitutive and nitrogen-regulated promoters of the petH gene encoding ferredoxin:NADP+ reductase in the heterocyst-forming cyanobacterium Anabaena sp.

Determination of the putative transcription start points of the petH gene encoding ferredoxin:NADP+ reductase in the heterocyst-forming cyanobacteria Anabaena sp. PCC 7119 and PCC 7120 showed that this gene is transcribed from two promoters, one constitutively used under different conditions of nitrogen nutrition and the other one used in cells subjected to nitrogen stepdown and in nitrogen-fixing filaments. The latter promoter, whose use was NtcA-dependent but HetR-independent, was functional in heterocysts. The N-control transcriptional regulator NtcA was observed to bind in vitro to this promoter. For the sake of comparison, the transcription start points of the nifHDK operon in strain PCC 7120 and binding of NtcA to the nifHDK promoter were also examined.

Anabaena↗
PubMed1997

Negatively charged anabaena flavodoxin residues (Asp144 and Glu145) are important for reconstitution of cytochrome P450 17alpha-hydroxylase activity.

Catalysis by microsomal cytochromes P450 requires the membrane-bound enzyme NADPH-cytochrome P450 reductase (P450 reductase), which transfers electrons to the P450 heme via a flavodoxin-like domain. Previously, we reported that Escherichia coli flavodoxin (Fld), a soluble electron transfer protein, directly interacts with bovine cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) and donates electrons to this enzyme when reconstituted with NADPH-ferredoxin (flavodoxin) reductase (FNR) (Jenkins, C. M., and Waterman, M. R. (1994) J. Biol. Chem. 269, 27401-27408). To investigate whether flavodoxins can serve as useful models of the analogous domain in P450 reductase, we have examined the FNR-Fld system from the cyanobacterium Anabaena. Mutagenesis of two acidic Anabaena Fld residues (D144A and E145A) significantly decreased flavodoxin-supported P450c17 progesterone 17alpha-hydroxylase activity. Specifically, D144A exhibited only 15% of the activity of wild-type Fld, whereas the adjacent mutation, E145A, caused a 40% loss in activity. P450-dependent hydrogen peroxide/superoxide production by wild-type FNR-Fld was measurably higher than that generated by FNR-D144A or FNR-E145A, indicating that the mutations do not lead to P450 heme-mediated electron uncoupling. Interestingly, the D144A and E145A mutants bind with equal or even greater affinity to P450c17 than wild-type Fld. Furthermore, these mutations (D144A and E145A) actually increased cytochrome c reductase activity (35 and 100% higher than wild type). Anabaena Fld residues Asp144 and Glu145 align closely with rat P450 reductase residue Asp208, which has been shown by mutagenesis to be important in electron transfer to P4502B1 but not to cytochrome c (Shen, A. L., and Kasper, C. B. (1995) J. Biol. Chem. 270, 27475-27480). Thus, these residues in flavodoxins and P450 reductase appear to have similar functions in P450 recognition and/or electron transfer, supporting the hypothesis that flavodoxins represent valid models for the FMN-binding domain of P450 reductase.

Amino Acid Sequence↗
PubMed1996

Overexpression in E. coli of the complete petH gene product from Anabaena: purification and properties of a 49 kDa ferredoxin-NADP+ reductase.

The complete petH gene product from Anabaena PCC 7119 has been overexpressed in E. coli and purified in order to determine the influence of the N-terminal extension on the interaction of ferredoxin-NADP+ reductase with its substrates. The intact 49 kDa FNR can be easily purified in a two-step procedure using batch extraction with DEAE-cellulose followed by Cibacron blue-Sepharose chromatography of the proteins unbound to DEAE. Isoelectric focusing of FNR shows several forms, with the major band at pH 6.26. The presence of the N-terminal extension increases the K(m) of FNR for NADPH by 4-fold and by 16.4-fold in the reduction reactions of DCPIP and cytochrome c. However, the K(m) for ferredoxin is 12-fold lower in the reaction catalyzed by the 49 kDa FNR than with the 36 kDa protein. This indicates that the presence of the third domain favours the interaction of FNR with ferredoxin, possibly due to the more positive net charge of the N-terminal extension. Comparable rate constants for both enzymes, were obtained for the photoreduction of NADP+ using photosynthetic membranes and also using rapid kinetic techniques. Slightly different ionic strength dependences of the rate constants were obtained, nevertheless, for both forms of the enzyme. These are a consequence of the structural differences that the proteins show at the N-terminal and of their effect on the interaction with ferredoxin.

Anabaena↗
PubMed1996

Expression of ferredoxin-NADP+ reductase in heterocysts from Anabaena sp.

The expression of ferredoxin-NADP+ reductase (FNR) from Anabaena sp. PCC 7119 in heterocysts and vegetative cells has been quantified. Specific reductase activity in heterocysts was approximately 10 times higher than in vegetative cells, corresponding to the increased FNR protein content. This was confirmed by immunoquantification of the FNR protein from whole filaments of Anabaena sp. PCC 7120 grown in media with and without combined nitrogen. Transcription of the petH gene was markedly enhanced in the absence of combined nitrogen. This suggests that the increased RNA level is mainly responsible for the up-regulation of FNR in heterocysts. As has been observed for nif genes, iron deficiency also increased transcription of petH. Characterization of the FNR purified from isolated heterocysts showed no detectable differences from the enzyme from vegetative cells. Although nitrogen stress was a key regulatory factor, localization of the petH gene in the genomic map of Anabaena PCC 7120 showed that this gene is not physically associated with the nif cluster.

Anabaena↗
PubMed1995

Site-specific mutagenesis demonstrates that the structural requirements for efficient electron transfer in Anabaena ferredoxin and flavodoxin are highly dependent on the reaction partner: kinetic studies with photosystem I, ferredoxin:NADP+ reductase, and cytochrome c.

Electron transfer reactions involving site-specific mutants of Anabaena ferredoxin (Fd) and flavodoxin (Fld) modified at surface residues close to the prosthetic groups, with photoexcited P700 in spinach photosystem I (PSI) particles, ferredoxin:NADP+ reductase (FNR), and horse cytochrome c (cytc), have been investigated by laser flash photolysis and stopped-flow spectrophotometry. Nonconservative mutations in Fd at F65 and E94, which have been shown to result in very large inhibitions of electron transfer to FNR, were found to yield wild-type behavior in reactions with PSI and cytc. In general, the effects of Fd mutagenesis on the PSI reactions were considerably smaller than those observed for the FNR reaction. In the case of Fld, mutagenesis was found to have only small effects on both the FNR and PSI reactions, although the specific sites whose mutation caused changes in electron transfer properties differed for the two systems. In contrast, several of the Fld mutants showed appreciably larger effects on the nonphysiological reaction with cytc. We conclude from these studies that the structural requirements for efficient electron transfer involving the Fd and Fld molecules differ, depending upon the reactant with which these redox proteins interact. This is consistent with the multiple roles that these proteins have in vivo in biological electron transfer and implies that different conserved residues in these proteins have evolved to satisfy varying requirements of particular reaction partners.

Anabaena↗
PubMed1994

Electron paramagnetic resonance as a tool for monitoring overexpression in Escherichia coli of fully functional flavodoxin.

Overexpression of foreign proteins in Escherichia coli is usually tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Western blot analysis. However, when metalloproteins of flavoenzymes are expressed, the correct assembly of the polypeptide chain to the cofactor cannot be verified using these methods. We have used EPR spectroscopy and Mancini tests to monitor the expression of holoflavodoxin from the cyanobacteria Anabaena in E. coli. Flavodoxin from Anabaena sp PCC 7119 was cloned in the plasmid pTrc 99b after the trc promotor, which is activated in the presence of isopropyl-beta-D-thiogalactoside. Two hours after induction, most of the recombinant apoflavodoxin is already synthesized. However, only 65% of this protein is assembled to flavin mononucleotide (FMN). Harvesting of the cells to obtain all the flavodoxin in the holo form must be carried out 10 h after induction. Addition of FMN to the culture media increases the synthesis of holoflavodoxin by about 17%. The presence of flavin adenine dinucleotide or riboflavin in the culture inhibits the accumulation of semiquinone flavodoxin in the cells.

Apoproteins↗
PubMed1994

Transcriptional and translational analysis of ferredoxin and flavodoxin under iron and nitrogen stress in Anabaena sp. strain PCC 7120.

In Anabaena sp. strain PCC 7120, vegetative cell ferredoxin synthesis under iron starvation was repressed 25-fold, whereas heterocyst ferredoxin synthesis decreased only 2.8-fold. Induction of flavodoxin under iron depletion was independent of the availability of combined nitrogen. Under iron stress but in the presence of combined nitrogen, fdxH and nifH genes were transcriptionally active; although excision of the 11-kb element seemed to be completed, nitrogenase activity and the fdxH gene product were not detectable.

Adaptation, Biological↗
PubMed1993

Homology of the N-terminal domain of the petH gene product from Anabaena sp. PCC 7119 to the CpcD phycobilisome linker polypeptide.

The complete nucleotide sequence of the petH gene encoding ferredoxin-NADP+ reductase from the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7119 has been determined. The encoded polypeptide is 136 amino acids longer than the enzyme obtained after purification to homogeneity. The extended N-terminal domain consists of 80 amino acids which shows homology to the CpcD phycobilisome linker polypeptide, through which FNR might be anchored to the thylakoid-bound phycobilisomes. A 56 amino acid interdomain fragment is found which could be a target for proteolysis.

Amino Acid Sequence↗
PubMed1991

Isolation and overexpression in Escherichia coli of the flavodoxin gene from Anabaena PCC 7119.

The gene coding for flavodoxin from Anabaena PCC 7119 was cloned by using the polymerase chain reaction (PCR). The gene is transcribed into a 1250-base transcript. The expression of the flavodoxin gene was analysed and found to be regulated at the transcriptional level by the availability of iron. The PCR-amplified gene was cloned into the expression vector pTrc 99b and expressed in Escherichia coli. High concentrations of flavodoxin were found (20% of total protein). The recombinant protein was purified from the cytosolic fraction of the cells and it exhibited properties identical with those of the wild-type Anabaena flavodoxin.

Amino Acid Sequence↗
PubMed1991

Light-dependent de-activation/re-activation of Anabaena variabilis ferredoxin: NADP+ reductase.

The activity of ferredoxin: NADP+ reductase (FNR) was found to decline to approximately 20% maximal levels with little or no loss in enzyme levels when cultures of the cyanobacterium Anabaena variabilis were maintained in the stationary phase of growth. Re-activation of enzyme activity occurred when cells were diluted into either fresh or re-utilized media and illuminated. This reversible de-activation/re-activation process was found, in vivo, to be dependent on the intensity of light illuminating the cells. The de-activated form of FNR was purified to homogeneity and exhibited the same molecular mass, isoelectric-focusing pattern and N-terminal amino acid sequence as the native form. Both de-activated and native FNR preparations each exhibited three reactive thiol groups on denaturation in urea; however, the rate of reaction with Ellman's reagent was much faster with the de-activated form than with the native form. Both preparations contain a single disulphide bond. Upon reduction of the disulphide bond in either form of the enzyme, the five reactive thiol groups exhibited identical reactivities in the presence of urea. Steady-state kinetic analysis of the de-activated form showed a marked increase in Km values for NADPH in diaphorase assays and an increase in Km for ferredoxin in the ferredoxin-mediated reduction of cytochrome c. No significant difference in kcat. was observed in comparison of the de-activated with the native form in any of the above assays; however, the de-activated form did exhibit a lower kcat. value in the transhydrogenase assay. The de-activated form of FNR bound ferredoxin with a 16-fold lower affinity than the native enzyme. These data suggest that the de-activation of FNR in vivo in response to low light intensity involves an alteration in protein structure, possibly via an intramolecular thiol disulphide interchange, which influences the interaction of the enzyme with its substrates.

Binding, Competitive↗