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R N Pau

Publications and source records attributed to R N Pau.

15 recordsLinked to original sources
PubMed1997

Characterisation of the molybdenum-responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli.

Molybdenum-dependent repression of transcription of the Escherichia coli modABCD operon, which encodes the high-affinity molybdate transporter, is mediated by the ModE protein. This regulatory protein was purified as an N-terminal His6-tagged derivative and characterised both with and without the N-terminal oligohistidine extension. Equilibrium centrifugation showed that ModE is at least a 57-kDa homodimer. Circular dichroism spectroscopy indicated that when molybdate or tungstate bind to ModE there is little change in its alpha-helical content, but a major change in the environment of tryptophan and tyrosine residues occurs. Addition of molybdate or tungstate to the protein results in almost 50% quenching of the fluorescence attributed to tryptophan. Titration of fluorescence quenching showed that two molecules of molybdenum bind to each dimer of ModE with a Kd of 0.8 microM. DNA mobility-shift assays showed that ModE requires molybdenum, or tungstate, to bind with high affinity (approximate Kd of 30 nM ModE) to the modABCD promoter region. In accord with ModE's role as a molybdenum-dependent transcriptional repressor, DNase I footprinting experiments showed that the ModE-molybdenum complex binds to a single 31-bp region around the transcription start of the modABCD promoter. This region contains a 6-base palindromic sequence CGTTAT-N12-ATAACG.

Bacterial Proteins↗
PubMed1996

The modE gene product mediates molybdenum-dependent expression of genes for the high-affinity molybdate transporter and modG in Azotobacter vinelandii.

The Azotobacter vinelandii mod locus, which is involved in high-affinity molybdate transport and the early event in Mo metabolism, consists of two divergently transcribed operons, modG and modEABC. modA, modB and modC encode the components of the high-affinity molybdate transporter, and modG encodes a Mo-binding protein. High concentrations of Mo repressed transcription of both operons. The modEABC operon was also repressed by tungstate and to a lesser extent by vanadate. modE, the first gene in the modEABC operon, controlled the Mo-dependent transcription of both operons. It was not involved in the metal regulation of alternative nitrogenase gene expression. Although a modE mutant constitutively expressed genes encoding the molybdate transporter, it had a reduced rate of Mo accumulation.

Azotobacter vinelandii↗
PubMed1996

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii.

In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo- and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15N2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo- and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative rho-independent transcription termination site has been identified 250 bp from the 3' end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo- and V-deficient conditions.

Acetylene↗
PubMed1995

Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii.

DNA sequencing of the region upstream from the Azotobacter vinelandii operon (modEABC) that contains genes for the molybdenum transport system revealed an open reading frame (modG) encoding a hypothetical 14-kDa protein. It consists of a tandem repeat of an approximately 65-amino-acid sequence that is homologous to Mop, a 7-kDa molybdopterin-binding protein of Clostridium pasteurianum. The tandem repeat is similar to the C-terminal half of the product of modE. The effects of mutations in the mod genes provide evidence for distinct high- and low-affinity Mo transport systems and for the involvement of the products of modE and modG in the processing of molybdate. modA, modB, and modC, which encode the component proteins of the high-affinity Mo transporter, are required for 99Mo accumulation and for the nitrate reductase activity of cells growing in medium with less than 10 microM Mo. The exchange of accumulated 99Mo with nonradioactive Mo depends on the presence of modA, which encodes the periplasmic molybdate-binding protein. 99Mo also exchanges with tungstate but not with vanadate or sulfate. modA, modB, and modC mutants exhibit nitrate reductase activity and 99Mo accumulation only when grown in more than 10 microM Mo, indicating that A. vinelandii also has a low-affinity Mo uptake system. The low-affinity system is not expressed in a modE mutant that synthesizes the high-affinity Mo transporter constitutively or in a spontaneous tungstate-tolerant mutant. Like the wild type, modG mutants only show nitrate reductase activity when grown in > 10 nM Mo. However, a modE modG double mutant exhibits maximal nitrate reductase activity at a 100-fold lower Mo concentration. This indicates that the products of both genes affect the supply of Mo but are not essential for nitrate reductase cofactor synthesis. However, nitrogenase-dependent growth in the presence or absence of Mo is severely impaired in the double mutant, indicating that the products of modE and modG may be involved in the early steps of nitrogenase cofactor biosynthesis in A. vinelandii.

Amino Acid Sequence↗
PubMed1993

Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor.

Nitrogenase-3 of Azotobacter vinelandii is synthesized under conditions of molybdenum and vanadium deficiency. The minimal metal requirement for its synthesis, and its metal content, indicated that the only transition metal in nitrogenase-3 was iron [Chisnell, Premakumar and Bishop (1988) J. Bacteriol. 170, 27-33; Pau, Mitchenall and Robson (1989) J. Bacteriol. 171, 124-129]. A new species of nitrogenase-3 has been purified from a strain of A. vinelandii (RP306) lacking structural genes for the Mo- and V-nitrogenases and containing a mutation which enables nitrogenase-3 to be synthesized in the presence of molybdenum. SDS/PAGE showed that component 1 contained a 15 kDa polypeptide which N-terminal amino acid sequence determination showed to be encoded by anfG. This confirms that nitrogenase-3, like V-nitrogenase, comprises three subunits. Preparations of the nitrogenase-3 from strain RP306 contained 24 Fe atoms and 1 Mo atom per molecule. Characterization of the cofactor centre of the enzyme by e.p.r. spectroscopy and an enzymic cofactor assay, together with stimulation of the growth of strain RP306 by Mo, showed that nitrogenase-3 can incorporate the Mo-nitrogenase cofactor (FeMoco) to form a functional enzyme. The specific activities (nmol of product produced/min per mg of protein) determined from activity titration curves were: under N2, NH3 formation 110, with concomitant H2 evolution of 220; under argon, H2 evolution 350; under 10% acetylene (C2H2) in argon, ethylene (C2H4) 58, ethane (C2H6) 26, and concomitant H2 evolution 226. The rate of formation of C2H6 was non-linear, and the C2H6/C2H4 ratio strongly dependent on the ratio of nitrogenase components.

Amino Acids↗
PubMed1993

Klebsiella pneumoniae nitrogenase MoFe protein: chymotryptic proteolysis affects function by limited cleavage of the beta-chain and provides high-specific-activity MoFe protein.

Proteinase treatment with chymotrypsin has been used to probe the structure of native Klebsiella pneumoniae nitrogenase MoFe protein (Kp1). Reaction with chymotrypsin did not bleach Kp1, suggesting that it did not destroy the metal centres, and the Mo and Fe contents of Kp1 were unchanged. High ratios of chymotrypsin to Kp1 (1:1 by mass) cleaved the beta-chain of Kp1 to give 44 and 14 kDa polypeptides, which N-terminal amino acid sequence analysis showed to be derived from cleavage at residue beta-Phe124. A mutant MoFe protein, Kp1Met-124, in which beta-Phe124 is replaced by methionine, was not cleaved by chymotrypsin. Under non-denaturing conditions, the 'nicked' beta-chain of the wild-type protein remained associated with the alpha-chain. The alpha-chain was not cleaved by the proteinase treatment. Fission of the wild-type beta-chain was accompanied by loss of enzyme activity, loss of intensity of the g = 3.7 e.p.r. signal derived from dithionite-reduced FeMoco and by changes in the visible spectrum. The e.p.r. spectra of potassium ferricyanide-oxidized native and digested Kp1 show differences in the signals between g = 1.6 and 2.0. After prolonged treatment, the final specific activity of Kp1 was about 25 +/- 5% of the initial activity. This corresponded to 25 +/- 5% of the beta-chain which was resistant to proteolytic action. Brief treatment of Kp1 with a lower concentration of chymotrypsin (chymotrypsin/Kp1 ratio = 1:10 by mass, for 10 min) preferentially cleaved high-molecular-mass polypeptides that routinely contaminate preparations of Kp1 prepared by standard procedures. Treatment with chymotrypsin followed by gel filtration to remove the proteinase and cleaved protein fragments can therefore be used to increase significantly the specific activity of Kp1 preparations and remove contaminating activities, such as the ATPase activity of myokinase.

Amino Acid Sequence↗
PubMed1993

Characterization of genes involved in molybdenum transport in Azotobacter vinelandii.

Expression of alternative nitrogenases in Azotobacter vinelandii is repressed by molybdenum. Two strains with Tn5 insertion mutations showed alternative nitrogenase-dependent diazotrophic growth in the presence of Mo. The mutations were in a region which contained four open reading frames (ORFs 1-4). The genetic structure and predicted products of ORFs 2, 3 and 4 are typical of the membrane-associated elements of the ATP-binding cassette (ABC) superfamily of transport systems. The products of ORF3 and ORF4 are homologous with the products of the Escherichia coli genes chlD and the partially sequenced chlJ, respectively, both of which are implicated in molybdenum transport. ORF1, which is in the relative position of bacterial permease genes commonly specifying periplasmic binding proteins, encodes a 29 kDa protein with a novel primary structure. It lacks a potential signal sequence, and its C-terminal half consists of a tandem repeat of a segment which is homologous with the M(r) 7 kDa molybdenum-pterin binding protein Mop from Clostridium pasteurianum. This suggests that a substituted pterin may be involved in the initial capture or early metabolism of molybdenum.

Amino Acid Sequence↗
PubMed1991

Transcriptional regulation by metals of structural genes for Azotobacter vinelandii nitrogenases.

Azotobacter vinelandii has three nitrogenases: a molybdenum (Mo) nitrogenase, a vanadium (V) nitrogenase, and a third nitrogenase (nitrogenase-3), which apparently lacks Mo and V. Mo represses synthesis of both V nitrogenase and nitrogenase-3, and in the absence of Mo, V represses synthesis of nitrogenase-3. We have investigated transcriptional regulation of the three nitrogenases by metals using Northern analysis and probes specific for transcripts of each of the three nitrogenases. Our results confirm that Mo is required for expression of the Mo nitrogenase structural genes (nifHDK), and substantiate the notion that Mo represses transcription of the structural genes for both V nitrogenase and nitrogenase-3. We show that repression by V of nitrogenase-3 is also effected at the level of transcription. Unexpectedly, V only represses transcription of the nitrogenase-3 structural genes (anfHDGK) if the V nitrogenase structural gene cluster vnfDGK is present. Further, deletion of nifHDK allows low expression of anfHDGK in the presence of Mo. Repression by Mo or V is independent of cofactor synthesis and therefore of enzyme activity.

Autoradiography↗
PubMed1990

Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii.

The nucleotide sequence (6,559 base pairs) of the genomic region containing the structural genes for nitrogenase 2 (V nitrogenase) from Azotobacter vinelandii was determined. The open reading frames present in this region are organized into two transcriptional units. One contains vnfH (encoding dinitrogenase reductase 2) and a ferredoxinlike open reading frame (Fd). The second one includes vnfD (encoding the alpha subunit of dinitrogenase 2), vnfG (encoding a product similar to the delta subunit of dinitrogenase 2 from A. chroococcum), and vnfK (encoding the beta subunit of dinitrogenase 2). The 5'-flanking regions of vnfH and vnfD contain sequences similar to ntrA-dependent promoters. This gene arrangement allows independent expression of vnfH-Fd and vnfDGK. Mutant strains (CA80 and CA11.80) carrying an insertion in vnfH are still able to synthesize the alpha and beta subunits of dinitrogenase 2 when grown in N-free, Mo-deficient, V-containing medium. A strain (RP1.11) carrying a deletion-plus-insertion mutation in the vnfDGK region produced only dinitrogenase reductase 2.

Amino Acid Sequence↗
PubMed1989

Structural genes for the vanadium nitrogenase from Azotobacter chroococcum.

Structural genes for the VFe-protein (Ac1V) of the vanadium nitrogenase from Azotobacter chroococcum were cloned and sequenced. The VFe-protein contains three subunit types with Mr of 53,793 (alpha), 52,724 (beta) and 13,274 (delta). alpha and beta subunits show 18 and 15% sequence identity respectively, with alpha and beta subunits of the MoFe-protein of A.chroococcum molybdenum nitrogenase. The genes for the three subunits vnfD (alpha), vnfG (delta) and vnfK (beta) are contiguous and form an operon whose transcription is repressed in response to ammonia. The Fe-protein component of the V-nitrogenase (Ac2V) is the product of nifH* that we have previously cloned and sequenced. This gene was located 2.5 kb upstream of vnfD. A deletion in the vnfD, G and K gene cluster prevents V-dependent nitrogen fixation. A strain defective in both V-nitrogenase and Mo-nitrogenase structural genes showed no residual nitrogen fixing capacity arguing against the presence of a third nitrogen fixation system in this organism.

Amino Acid Sequence↗
PubMed1989

Nitrogenases without molybdenum.

For 50 years molybdenum had been considered to have an indispensable catalytic function for nitrogen fixation. Two nitrogenases recently isolated from the bacterium Azotobacter have changed this view. One is a vanadium-containing enzyme and the other lacks both molybdenum and vanadium. Similar nitrogenases may occur in other nitrogen-fixing organisms.

Amino Acid Sequence↗
PubMed1989

Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium.

We have constructed a strain of Azotobacter vinelandii which has deletions in the genes for both the molybdenum (Mo) and vanadium (V) nitrogenases. This strain fixed nitrogen in medium that did not contain Mo or V. Growth and nitrogenase activity were inhibited by Mo and V. In highly purified medium, growth was limited by iron. Addition of other metals (Co, Cr, Cu, Mn, Ni, Re, Ti, W, and Zn) did not stimulate growth. Like the V-nitrogenase, the nitrogenase synthesized by the double deletion strain reduced acetylene to both ethylene and ethane (C2H6/C2H4 ratio, 0.046). There was an approximately 10-fold increase in ethane production when Mo was added to the deletion strain grown in medium lacking Mo and V. This change in reactivity may be due to the incorporation of an Mo-containing cofactor into the nitrogenase synthesized by the double-deletion strain. A strain synthesizing the V-nitrogenase did not show a similar increase in ethane production. The growth characteristics of the double-deletion strain, together with the metal composition reported for a nitrogenase isolated from a tungstate-tolerant strain lacking genes for the molydenum enzyme grown in the absence of Mo and V (J. R. Chisnell, R. Premakumar, and P. E. Bishop, J. Bacteriol. 170:27-33, 1988) show that A. vinelandii can synthesize a nitrogenase which lacks both Mo and V. Reduction of dinitrogen by nitrogenase can therefore occur at a center lacking both these metals.

Azotobacter↗
PubMed1975

The hydroxylation of tyrosine by an enzyme from third-instar larvae of the blowfly Calliphora erythrocephala.

1. Two pro-(phenol oxidase) were distinguished when the blood of late-third-instar larvae of Calliphora erythrocephala was electrophoresed in polyacrylamide gels with Tris-glycine buffer, pH 8.3. One pro-(phenol oxidase), after activation by an enzyme readily catalyses the oxidation of both L-tyrosine and L-3,4-dihydroxyphenylalanine (L-dopa). The second enzyme catalyses the oxidation of L-dopa but not of L-tyrosoine. 2. One of the pro-(phenol oxidases) was purified over 2000-fold from homogenates of whole larvae. This enzyme, after activation, catalyses the oxidation of both dopa and tyrosine. On electrophoresis in polyacrylamide gels with Tris-glycine buffer, pH 8.3, it has the same mobility as the enzyme in the blood which catalyses the oxidation of both tyrosine and dopa. 3. The pro-(phenol oxidase)-activating enzyme was purified over 100-fold from homogenates of whole larvae. 4. The oxidation of L-tyrosine, in the presence of the activated purified phenol oxidase, reached a steady maximum rate after a lag period that was directly related to tyrosine concentration and inversely related to enzyme concentration. 5. The effect of the addition of electron donors on the lag period was studied. Dopa, dopamine (3,4-dihydroxyphenethylamine) and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine are the most effective hydrogen donors. 3,4-Dihydroxybenzoic acid, the oxidation of which was not catalysed by the activated pro-(phenol oxidase), did not affect the lag period.

Animals↗
PubMed1975

The isolation of an o-diphenal oxidase from third-instar larvae of the blowfly Calliphora erythrocephala.

1. The isolation of an o-diphenol oxidase from an acetone-dried powder of late-third-instar larvae of Calliphora erythrocephala was investigated. An insoluble and micro-crystalline fraction containing the enzyme activity was obtained after fractionating extracts of the acetone-dried powder with (NH4)2SO4 and acetone. 2. This fraction can be solubilized in 0.1% sodium dodecyl sulphate without loss of activity. 3. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate shows that the o-diphenol oxidase is a minor component of the extracts from the acetone-dried powder. 4. The o-diphenol oxidase was purified by zonal centrifugation on a sucrose density gradient in the presence of sodium dodecyl sulphate. 5. The amino acid composition of the purified enzyme resembles that of some other o-diphenol oxidases. 6. The subunit composition of the o-diphenol oxidase is discussed.

Amino Acids↗