Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage

Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-(32)P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-(14)C]NAD individually upon UV irradiation, but most (14)C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-(14)C]NAD suggested that Arg 101 is not absolutely required for NAD binding.     

ADP-Ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The structure of the dinitrogenase reductase from Azotobacter vinelandii is known. In this study, mutant forms of dinitrogenase reductase from A. vinelandii that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum. R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction. Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe(4)S(4) cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT. Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT. Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073-2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.     

Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum

The enzyme that catalyzes the ADP-ribosylation and concomitant inactivation of dinitrogenase reductase in Rhodospirillum rubrum has been purified greater than 19,000-fold to near homogeneity. We propose dinitrogenase reductase ADP-ribosyltransferase (DRAT) as the working name for the enzyme. DRAT activity is stabilized by NaCl and ADP. The enzyme is a monomer with a molecular mass of 30 kDa and is a different polypeptide than dinitrogenase reductase activating glycohydrolase. NAD (Km = 2 mM), etheno-NAD, nicotinamide hypoxanthine dinucleotide, and nicotinamide guanine dinucleotide will serve as donor molecules in DRAT-catalyzed ADP-ribosylation reaction, and dinitrogenase reductases from R. rubrum, Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianium will serve as acceptors. No other proteins or small molecules, including water, have been found to be effective as acceptors. Nicotinamide is released stoichiometrically with formation of the ADP-ribosylated product. DRAT is inhibited by NaCl and has maximal activity at a pH of 7.0.     

Effect of nucleotides on the activity of dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum

The mechanism by which MgADP stimulates the activity of dinitrogenase reductase ADP-ribosyltransferase (DRAT) has been examined by using dinitrogenase reductases from Rhodospirillum rubrum, Klebsiella pneumoniae, and Azotobacter vinelandii as acceptor substrates. In the presence of 0.2 mM NAD, maximal rates of ADP-ribosylation of all three acceptors were observed at an ADP concentration of 150 microM; in the absence of added ADP, DRAT activity with the dinitrogenase reductases from R. rubrum and K. pneumoniae was less than 5% of the maximal rate, but the A. vinelandii protein was ADP-ribosylated at 40% of the maximal rate. Of eight dinucleotides tested, only ADP, 2'-deoxy-ADP, and ADP-beta S served as activators of the DRAT reaction; ADP, 2'-deoxy-ADP, and ADP-beta S were also the only dinucleotides found which inhibited acetylene reduction activity by dinitrogenase reductase. The dinucleotide specificities for both DRAT activation and acetylene reduction inhibition were the same for all three dinitrogenase reductases. In the DRAT reaction with the dinitrogenase reductases from K. pneumoniae and A. vinelandii, the Km for NAD was 30-fold higher in the absence of ADP than in its presence; the Km for NAD with the R. rubrum acceptor was not measurable. In the presence of saturating ADP, ADP-ribosylation of dinitrogenase reductase from R. rubrum was inhibited 63% by 1.5 mM ATP. It is concluded that MgADP stimulates DRAT activity by lowering the Km for NAD and that MgADP exerts its effect by binding to dinitrogenase reductase. MgATP inhibits DRAT activity by competing with MgADP for binding to dinitrogenase reductase.     

NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii

Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversibly regulated by ADP-ribosylation of a specific arginine residue of dinitrogenase reductase based on the cellular nitrogen or energy status. In this paper, we have investigated the ability of nicotinamide adenine dinucleotide, NAD (the physiological ADP-ribose donor), and its analogs to support covalent modification of dinitrogenase reductase in vitro. R. rubrum dinitrogenase reductase can be modified by DRAT in the presence of 2 mM NAD, but not with 2 mM nicotinamide mononucleotide (NMN) or nicotinamide adenine dinucleotide phosphate (NADP). We also found that the apo- and the all-ferrous forms of R. rubrum dinitrogenase reductase are not substrates for covalent modification. In contrast, Azotobacter vinelandii dinitrogenase reductase can be modified by the dinitrogenase reductase ADP-ribosyl transferase (DRAT) in vitro in the presence of either 2 mM NAD, NMN or NADP as nucleotide donors. We found that: (1) a simple ribose sugar in the modification site of the A. vinelandii dinitrogenase reductase is sufficient to inactivate the enzyme, (2) phosphoADP-ribose is the modifying unit in the NADP-modified enzyme, and (3) the NMN-modified enzyme carries two ribose-phosphate units in one modification site. This is the first report of NADP- or NMN-dependent modification of a target protein by an ADP-ribosyl transferase.     

Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase.

Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation.     

Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense.

Reversible ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system, has been characterized in both Rhodospirillum rubrum and Azospirillum brasilense. Although the general functions of DRAT and DRAG are very similar in these two organisms, there are a number of interesting differences, e.g., in the timing and extent of the regulatory response to different stimuli. In this work, the basis of these differences has been studied by the heterologous expression of either draTG or nifH from A. brasilense in R. rubrum mutants that lack these genes, as well as the expression of draTG from R. rubrum in an A. brasilense draTG mutant. In general, these hybrid strains respond to stimuli in a manner similar to that of the wild-type parent of the recipient strain rather than the wild-type source of the introduced genes. These results suggest that the differences seen in the regulatory response in these organisms are not primarily a result of different properties of DRAT, DRAG, or dinitrogenase reductase. Instead, the differences are likely the result of different signal pathways that regulate DRAG and DRAT activities in these two organisms. Our results also suggest that draT and draG are cotranscribed in A. brasilense.     

Functional expression of a Rhodospirillum rubrum gene encoding dinitrogenase reductase ADP-ribosyltransferase in enteric bacteria

The function of the cloned draT gene of Rhodospirillum rubrum was studied by placing it under the control of the tac promoter in the vector, pKK223-3. After induction with isopropyl-beta-D-thiogalactopyranoside, dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in crude extracts of the heterologous hosts Escherichia coli and Klebsiella pneumoniae. In addition, the expression of draT produced a Nif- phenotype in the otherwise wild-type K. pneumoniae strains, the result of the ADP-ribosylation of accumulated dinitrogenase reductase (DR). DR from a nifF- background was also susceptible to ADP-ribosylation, indicating that the oxidized form of DR will serve as a substrate for DRAT in vivo. A mutation that changes the Arg-101 residue of DR, the ADP-ribose attaching site, eliminates the ADP-ribosylation of DR in vivo, confirming the necessity of this residue for modification.     

Effect of P(II) and its homolog GlnK on reversible ADP-ribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase regulatory system in Klebsiella pneumoniae

Reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase (DRAT-DRAG) regulatory system, has been characterized in Rhodospirillum rubrum and other nitrogen-fixing bacteria. To investigate the mechanisms for the regulation of DRAT and DRAG activities, we studied the heterologous expression of R. rubrum draTG in Klebsiella pneumoniae glnB and glnK mutants. In K. pneumoniae wild type, the regulation of both DRAT and DRAG activity appears to be comparable to that seen in R. rubrum. However, the regulation of both DRAT and DRAG activities is altered in a glnB background. Some DRAT escapes regulation and becomes active under N-limiting conditions. The regulation of DRAG activity is also altered in a glnB mutant, with DRAG being inactivated more slowly in response to NH4+ treatment than is seen in wild type, resulting in a high residual nitrogenase activity. In a glnK background, the regulation of DRAT activity is similar to that seen in wild type. However, the regulation of DRAG activity is completely abolished in the glnK mutant; DRAG remains active even after NH4+ addition, so there is no loss of nitrogenase activity. The results with this heterologous expression system have implications for DRAT-DRAG regulation in R. rubrum.     

Substitution of histidine for arginine-101 of dinitrogenase reductase disrupts electron transfer to dinitrogenase

Dinitrogenase reductase from Klebsiella pneumoniae strain UN1041 has a histidine residue substituted for arginine at position 101. The mutant dinitrogenase reductase was purified and characterized in order to determine the importance of arginine-101 in the interaction between dinitrogenase and dinitrogenase reductase during electron transfer. Purified dinitrogenase reductase from UN1041 is a dimer of 67 kDa, contains a functional 4Fe-4S cluster, undergoes a MgATP-dependent conformational change, and is competent for ATP hydrolysis uncoupled from substrate reduction in the presence of dinitrogenase. However, the mutant protein is unable to support the reduction of protons or acetylene by dinitrogenase. A 100-fold molar excess of Kp2 from UN1041 does not inhibit electron transfer from wild-type dinitrogenase reductase to dinitrogenase. It is concluded that the interaction of dinitrogenase reductase with dinitrogenase during reductant-independent ATP hydrolysis is different than the interaction between the two proteins during electron transfer; the substitution of histidine for arginine at position 101 disrupts only the latter interaction. The same conclusions are reached using wild-type dinitrogenase reductase which has been ADP-ribosylated at arginine-101.     

Regulation of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase by a redox-dependent conformational change of nitrogenase Fe protein

The nitrogenase-regulating enzymes dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG), from Rhodospirillum rubrum, were shown to be sensitive to the redox status of the [Fe(4)S(4)](1+/2+) cluster of nitrogenase Fe protein from R. rubrum or Azotobacter vinelandii. DRAG had <2% activity with oxidized R. rubrum Fe protein relative to activity with reduced Fe protein. The activity of DRAG with oxygen-denatured Fe protein or a low molecular weight substrate, N(alpha)-dansyl-N(omega)-(1,N(6)-etheno-ADP-ribosyl)-arginine methyl ester, was independent of redox potential. The redox midpoint potential of DRAG activation of Fe protein was -430 mV versus standard hydrogen electrode, coinciding with the midpoint potential of the [Fe(4)S(4)] cluster from R. rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low (<20%) activity with 87% reduced R. rubrum Fe protein relative to activity with fully oxidized Fe protein. A mutant of R. rubrum in which the rate of oxidation of Fe protein was substantially decreased had a markedly slower rate of ADP-ribosylation in vivo in response to 10 mM NH(4)Cl or darkness stimulus. It is concluded that the redox state of Fe protein plays a significant role in regulation of the activities of DRAT and DRAG in vivo.     

Characterization of altered regulation variants of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum

In Rhodospirillum rubrum, nitrogenase activity is subject to posttranslational regulation through the adenosine diphosphate (ADP)-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG). To study the posttranslational regulation of DRAG, its gene was mutagenized and colonies screened for altered DRAG regulation. Three different mutants were found and the DRAG variants displayed different biochemical properties including an altered affinity for divalent metal ions. Taken together, the results suggest that the site involved in regulation is physically near the metal binding site of DRAG.     

Correlation of activity regulation and substrate recognition of the ADP-ribosyltransferase that regulates nitrogenase activity in Rhodospirillum rubrum

In Rhodospirillum rubrum, nitrogenase activity is regulated posttranslationally through the ADP-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered both in the posttranslational regulation of DRAT activity and in the ability to recognize variants of dinitrogenase reductase have been found. This correlation suggests that these two properties are biochemically connected.     

N-glycohydrolysis of adenosine diphosphoribosyl arginine linkages by dinitrogenase reductase activating glycohydrolase (activating enzyme) from Rhodospirillum rubrum

The reaction catalyzed by the activating enzyme for dinitrogenase reductase from Rhodospirillum rubrum has been studied using an ADP-ribosyl hexapeptide, obtained from proteolysis of inactive dinitrogenase reductase, and synthetic analogs such as N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester. The activating enzyme catalyzed N-glycohydrolysis of the ribosyl-guanidinium linkage releasing ADP-ribose and regenerating an unmodified arginyl guanidinium group. Optimal glycohydrolysis of the low molecular weight substrates occurred at pH 6.6 and required 1 mM MnCl2, but did not require ATP. The ADP-ribosyl hexapeptide (Km 11 microM), N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester (Km 12 microM), N alpha-dansyl-N omega-ADP-ribosylarginine (Km 12 microM), N alpha-dansyl-N omega-1,N6-etheno-ADP-ribosylarginine methyl ester (Km 11 microM), and N alpha-dansyl-N omega-GDP-ribosylarginine methyl ester (Km 11 microM) were comparable substrates. N omega-ADP-ribosylarginine (Km 2 mM) was a poor substrate, and the activating enzyme did not catalyze N-glycohydrolysis of N alpha-dansyl-N omega-5'-phosphoribosylarginine methyl ester or N alpha-dansyl-N omega-ribosylarginine methyl ester. 13C NMR of N alpha-tosyl-N omega-ADP-ribosylarginine methyl ester established that the activating enzyme specifically hydrolyzed the alpha-ribosyl-guanidinium linkage. The beta-linked anomer was hydrolyzed only after anomerization to the alpha configuration. We recommend [arginine(N omega-ADP-alpha-ribose)]dinitrogenase reductase N-glycohydrolase (dinitrogenase reductase activating) and dinitrogenase reductase activating glycohydrolase as the systematic and working names for the activating enzyme.     

Posttranslational modification of dinitrogenase reductase in <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> treated with fluoroacetate

Nitrogen fixation is one of the major biogeochemical contributions carried out by diazotrophic microorganisms. The goal of this research is study of posttranslational modification of dinitrogenase reductase (Fe protein), the involvement of malate and pyruvate in generation of reductant in Rhodospirillum rubrum. A procedure for the isolation of the Fe protein from cell extracts was developed and used to monitor the modification of the Fe protein in vivo. The subunit pattern of the isolated the Fe protein after sodium dodecyl sulfate–polyacrylamide gel electrophoresis was assayed by Western blot analysis. Whole-cell nitrogenase activity was also monitored during the Fe protein modification by gas chromatograpy, using the acetylene reduction assay. It has been shown, that the addition of fluoroacetate, ammonia and darkness resulted in the loss of whole-cell nitrogenase activity and the in vivo modification of the Fe protein. For fluoroacetate, ammonia and darkness, the rate of loss of nitrogenase activity was similar to that for the Fe protein modification. The addition of NADH and reillumination of a culture incubated in the dark resulted in the rapid restoration of nitrogenase activity and the demodification of the Fe protein. Fluoroacetate inhibited the nitrogenase activity of R. rubrum and resulted in the modification of the Fe protein in cells, grown on pyruvate or malate as the endogeneous electron source. The nitrogenase activity in draTG mutant (lacking DRAT/DRAG system) decreased after the addition of fluoroacetate, but the Fe protein remained completely unmodified. The results showed that the reduced state of cell, posttranslational modifications of the Fe protein and the DRAT/DRAG system are important for nitrogenase activity and the regulation of nitrogen fixation.     

Borate inhibits activation of inactive dinitrogenase reductase from Rhodospirillum rubrum

Borate and aminophenylboronic acid were tested as inhibitors of activation of inactive dinitrogenase reductase from Rhodospirillum rubrum. Inhibition was specific for activation because activity of the active form of the enzyme was not inhibited. Inhibition showed the pH-dependence expected if borate inhibits by binding to cis-hydroxy groups of the modifying group found on the inactive enzyme.     

Proteolytic degradation of dinitrogenase reductase from Anabaena variabilis (ATCC 29413) as a consequence of ATP depletion and impact of oxygen.

Both components of nitrogenase, dinitrogenase and dinitrogenase reductase, are rapidly inactivated by oxygen. To investigate the proteolytic degradation of dinitrogenase reductase irreversibly destroyed by high oxygen concentrations, we carried out in vitro experiments with heterocyst extracts from Anabaena variabilis ATCC 29413. The results indicate a direct dependence of degradation on the applied oxygen concentration. Although the degrees of degradation were similar for both the modified and unmodified subunits of dinitrogenase reductase, there was a significant difference with respect to the cleavage products observed. The pattern of effective protease inhibitors suggests the involvement of serine proteases with chymotrypsin- and trypsin-like specificity. A protective effect was obtained by saturation of the nucleotide binding sites of dinitrogenase reductase with either ATP or ADP. As shown by gel filtration experiments, the adenylates prevented the nitrogenase subunits from extensive noncovalent aggregation, which is usually considered evidence for a denaturing process. The in vitro degradation of dinitrogenase reductase is discussed in connection with previous reports on degradation of nitrogenase in cyanobacteria under oxygen stress and/or starvation.     

Cloning and expression of draTG genes from Azospirillum lipoferum

A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum. As in R. rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes. Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system.     

Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium.

Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.