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Journal of Bacteriology, May 2000, p. 2597-2603, Vol. 182, No. 9
Department of
Biochemistry1 and Center for the Study
of Nitrogen Fixation,2 College of
Agricultural and Life Sciences, University of Wisconsin
Received 12 October 1999/Accepted 26 January 2000
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
Fe4S4 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.
Nitrogenase activity in
Rhodospirillum rubrum is regulated by reversible
ADP-ribosylation of dinitrogenase reductase at Arg 100. Dinitrogenase
reductase ADP-ribosyltransferase (DRAT) catalyzes the transfer of
the ADP-ribose moiety from NAD to Arg 100, rendering dinitrogenase
reductase inactive. The enzyme is reactivated upon removal of
ADP-ribose by dinitrogenase reductase-activating glycohydrolase (DRAG).
DRAT is very specific for native dinitrogenase reductase as a
substrate. No other acceptor molecules have been found, although several have been tested; these include oxygen-denatured dinitrogenase reductase, arginine, dansylarginine, and a hexapeptide of
dinitrogenase reductase containing Arg 100, the site of
ADP-ribosylation (14, 16). DRAT can modify
dinitrogenase reductases from Azotobacter vinelandii,
Klebsiella pneumoniae, and Clostridium
pasteurianum, although these organisms do not contain endogenous
ADP-ribosylation systems (14).
Seefeldt and coworkers have created and characterized
several site specifically altered A. vinelandii
dinitrogenase reductases which have decreased ability relative to
wild-type dinitrogenase reductase to support substrate reduction;
these researchers thereby have identified regions of dinitrogenase
reductase important in the transfer of electrons to dinitrogenase
(11, 20, 21). Figure 1 shows
the structure of dinitrogenase reductase (4) and the
positions of the residues described in this paper. Characterization of
the altered dinitrogenase reductase in which Asp 129 was replaced with
Glu suggests that Asp 129 is involved in MgATP hydrolysis (11), which is coupled to the transfer of electrons from
dinitrogenase reductase to dinitrogenase. Studies also have shown that
both Arg 140 and Lys 143 are important for the docking of dinitrogenase reductase and dinitrogenase (22). Seefeldt and coworkers
have also characterized an altered dinitrogenase reductase in which Leu
127 was deleted (Leu127
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
ADP-Ribosylation of Variants of Azotobacter vinelandii
Dinitrogenase Reductase by Rhodospirillum rubrum
Dinitrogenase Reductase ADP-Ribosyltransferase
Madison,
Madison, Wisconsin 53706-1544, and Department of Chemistry
and Biochemistry, Utah State University, Logan, Utah
843323
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
) (10, 21). Leu 127 is located between Asp 125, which is located in the nucleotide binding site and
interacts with the Mg2+ ion associated with the nucleotide
(23), and Cys 132, which is a ligand for the
Fe4S4 cluster (7). The
Fe4S4 cluster of the nucleotide-free Leu127
dinitrogenase reductase has chemical properties similar to those of the
Fe4S4 cluster of the MgATP-bound wild-type
dinitrogenase reductase. Leu127 dinitrogenase reductase also binds
tightly to dinitrogenase in the presence and absence of MgATP.
View larger version (69K):
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FIG. 1.
Crystal structure of A. vinelandii
dinitrogenase reductase as determined by Georgiadis et al.
(4). The following residues are highlighted: Arg 100, Arg
140, Phe 135, Lys 143, Asp 129, Asp 43, and Leu 127 and the iron atoms
of the Fe4S4 cluster. This structure is based
on the form of A. vinelandii dinitrogenase reductase with
one molecule of ADP bound.
In this work, we used several well-characterized altered forms of dinitrogenase reductase to further define the structural properties required for dinitrogenase reductase to interact with DRAT and also for dinitrogenase reductase to be a substrate for ADP-ribosylation.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Purification of the altered dinitrogenase reductases. Purification of the altered dinitrogenase reductases was performed by Lance Seefeldt and coworkers at Utah State University as previously described (11, 20-22). All of these proteins are "native" in that they are dimers with Fe4S4 clusters with a normal electron paramagnetic resonance signal.
Purification of DRAT. DRAT was purified from R. rubrum strain UR356 as previously described (5).
ADP-ribosylation of the altered dinitrogenase reductases. ADP-ribosylation reactions were performed with microcentrifuge tubes that had been placed inside 9-ml vials that were made anaerobic by repeated evacuation and flushing with nitrogen. A solution containing 100 mM dithionite was added to the vial on the outside of the microcentrifuge tube (16). This procedure creates an anaerobic environment that permits oxygen to be scavenged with a minimum amount of dithionite in the reaction mixture (dithionite reduces NAD, the ADP-ribose donor). Dinitrogenase reductase was incubated in a 40-µl reaction mixture containing 9.0 µg of purified protein, 15 mM HEPES (pH 7.6), 0.1 mM sodium dithionite, 2 mM NAD, 1.25 mM MgADP, and 1.5 µg of purified DRAT at 30°C for 3 min. (MgADP stimulates the ADP-ribosylation of A. vinelandii dinitrogenase reductase [15]). The reaction was stopped by the addition of 40 µl of sodium dodecyl sulfate (SDS) buffer containing 130 mM Tris (pH 6.8), 4.2% (wt/vol) SDS, 20% (vol/vol) glycerol, 0.003% (wt/vol) bromphenol blue, and 10% (vol/vol) 2-mercaptoethanol (added fresh), and the mixture was boiled for 1 min. After this step, the reaction mixture was diluted 10-fold, and 5.0 µl was loaded onto a polyacrylamide gel (10% [wt/vol] total acrylamide; ratio of acrylamide to bisacrylamide, 172:1) to resolve the modified and unmodified subunits of dinitrogenase reductase by electrophoresis (9). The separated proteins were transferred to a nitrocellulose membrane, which was then incubated with polyclonal antibodies against dinitrogenase reductase (1:5,000, 1 h). The position of dinitrogenase reductase on the blot was visualized by chemiluminescence (Amersham), and the proteins were quantitated by densitometry scanning. Some mutant forms of dinitrogenase reductase migrate anomolously on SDS gels.
Cross-linking of the altered dinitrogenase reductases and DRAT. Cross-linking reactions were also performed with microcentrifuge tubes prepared as described above (6). Dinitrogenase reductase was incubated in a 40-µl reaction mixture containing 9.0 µg of purified protein, 15 mM HEPES (pH 7.6), 0.1 mM sodium dithionite, 2 mM NAD, 1.25 mM MgADP, 3.0 µg of purified DRAT, and 5 mM (1-ethyl-3-(3-dimethylaminopropyl)-carbodimide (EDC). The cross-linking reaction was incubated at 30°C for 3 min and then stopped by the addition of 40 µl of SDS buffer. After the mixture was boiled for 1 min, 5.0-µl reaction samples were loaded onto a gel as described above. The separated proteins were electrophoretically transferred to a nitrocellulose membrane, which was then incubated with polyclonal antibodies against DRAT (1:1,000, 1 h). The blot was then incubated with anti-rabbit immunoglobulin G-horseradish peroxidase conjugate (1:3,000, 40 min). The position of the DRAT-dinitrogenase reductase complex on the blot was visualized by chemiluminescence (Amersham), and quantitation was done by densitometry scanning.
Preparation of apo-dinitrogenase reductase.
Apo-dinitrogenase reductase for this study was prepared by Priya
Rangaraj as previously described (13, 24). Briefly, 2.0 mg
of purified dinitrogenase reductase was incubated with 20 µmol of
, '-dipyridyl in the presence of 2.5 mM MgATP and 2 mM sodium dithionite. After incubation at 25°C for 30 min, apo-dinitrogenase reductase was passed over a Sephadex G-25 column (1 by 10 cm) equilibrated with 25 mM Tris-HCl (pH 7.4) and 2.0 mM sodium dithionite to remove ,'-dipyridyl. To confirm the complete conversion of holo-Fe protein to apo-Fe protein, the apo-Fe protein preparation was
coupled to MoFe protein in an in vitro acetylene reduction assay and
shown to contain no substrate reduction ability. However, the prepared
apo-dinitrogenase reductase was still active in its ability to support
iron-molybdenum cofactor synthesis (19). Zheng et al. have
demonstrated the ability of apo-dinitrogenase reductase prepared in
this manner to be reconstituted with a normal Fe4S4 cluster by NifS (24).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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The following altered A. vinelandii dinitrogenase
reductases, which have a decreased ability, relative to wild-type
dinitrogenase reductase, to support substrate reduction, were
characterized for their ability to be substrates for DRAT-catalyzed
ADP-ribosylation: R140Q, K143Q, F135Y, D43N, D129E, and L127. A
decrease in dinitrogenase reductase ADP-ribosylation could be due to a
decreased ability of the altered dinitrogenase reductase to form a
complex with DRAT; therefore, the interaction between these altered
dinitrogenase reductases and DRAT was studied via chemical
cross-linking of the two proteins. Table
1 depicts the previously described
properties of these altered dinitrogenase reductases and the data
obtained here. Figure 1 shows the crystal structure of A. vinelandii dinitrogenase reductase, with the above-mentioned
residues highlighted.
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R140Q dinitrogenase reductase.
Replacement of R140 with a Q
residue renders dinitrogenase unable to be ADP-ribosylated. R140Q
dinitrogenase reductase was incubated in an ADP-ribosylation reaction
as described in Materials and Methods. Analysis of this reaction on
dinitrogenase reductase immunoblots showed that R140Q dinitrogenase
reductase was not ADP-ribosylated (Fig.
2, lane 2b). Reaction conditions were
such that 78% of wild-type dinitrogenase reductase was ADP-ribosylated (Fig. 2, lane 1b). (One hundred percent ADP-ribosylation is represented by equal quantities of modified and unmodified dinitrogenase reductase subunits.) ADP-ribosylation of R140Q dinitrogenase reductase was not
observed even when the incubation period was increased to 20 min (data
not shown). These data indicate that the R140Q substitution disrupts
the ability of dinitrogenase reductase to be a substrate for
ADP-ribosylation.
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The Fe4S4 cluster region of dinitrogenase reductase is important for ADP-ribosylation. We also wanted to test if the conformation of the Fe4S4 cluster region is important for maintaining the proper orientation of Arg 100 such that ADP-ribosylation of dinitrogenase reductase can occur. Crystallographic data show that Phe 135 is located at the interface between the dinitrogenase reductase subunits near the Fe4S4 cluster (4). The F135Y dinitrogenase reductase protein contains an intact Fe4S4 cluster, as determined by electron paramagnetic resonance studies; however, circular dichroism studies show that the environment of the Fe4S4 cluster in this protein is different from that in wild-type dinitrogenase reductase (20). Therefore, F135Y dinitrogenase reductase was analyzed for its ability to be ADP-ribosylated. Only 6% of F135Y dinitrogenase reductase was ADP-ribosylated when incubated with DRAT and NAD under the stated reaction conditions (Fig. 2, lane 4b), in which 78% of wild-type dinitrogenase reductase was ADP-ribosylated (Fig. 2, lane 1b). This result indicates that F135Y dinitrogenase reductase is not an effective substrate for ADP-ribosylation.
Cross-linking studies show that the extent of formation of a cross-linkable complex between DRAT and F135Y dinitrogenase reductase is approximately 40 times lower than that between DRAT and wild-type dinitrogenase reductase under these conditions (Fig. 3, lane 4). This result indicates that the F135Y mutation changes the conformation of dinitrogenase reductase in such a way as to decrease the ability of DRAT to cross-link with dinitrogenase reductase. There are two possible explanations for this decrease in cross-linking: (i) F135Y dinitrogenase reductase may not interact with DRAT, and thus no cross-linkable complex is formed between the two proteins, or (ii) F135Y dinitrogenase reductase interacts with DRAT but in a conformation such that cross-linking between the two proteins does not occur. If DRAT and F135Y dinitrogenase reductase do still interact, it is in such a way that an unproductive complex forms and ADP-ribosylation of F135Y dinitrogenase reductase does not occur.The Fe4S4 cluster is required for
ADP-ribosylation and cross-linking with DRAT.
Consistent with
these results is the inability of wild-type apo-dinitrogenase reductase
to be ADP-ribosylated in the DRAT-catalyzed ADP-ribosylation reaction
(Fig. 4A, lane 2). The
Fe4S4 cluster of wild-type A. vinelandii dinitrogenase reductase was removed as described in
Materials and Methods by treatment of dinitrogenase reductase with the
chelator ,'-dipyridyl (14, 24). Apo-dinitrogenase reductase also did not form a cross-linkable complex with DRAT when
added to the previously described cross-linking reaction mixture (Fig.
4B, lane 2). These results show that removal of the
Fe4S4 cluster does change the conformation of
dinitrogenase reductase such that apo-dinitrogenase reductase does not
form a productive complex with DRAT and therefore is not a substrate for ADP-ribosylation. Apo-dinitrogenase reductase is effective in its
role in iron-molybdenum cofactor synthesis (18).
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ADP-ribosylation of dinitrogenase reductase altered in the
nucleotide binding region.
The ADP-ribosylation of A. vinelandii dinitrogenase reductase is stimulated by the presence
of MgADP. Previous data suggested that this effect is due to the
binding of MgADP to dinitrogenase reductase (15), which
contains two nucleotide binding sites per dimer, rather than to DRAT
itself. These nucleotide binding sites have been extensively studied
for their role in nitrogenase substrate reduction. The nucleotide
binding sites are located in the cleft between the dinitrogenase
reductase subunits and are oriented with the triphosphates toward the
Fe4S4 cluster. The role of nucleotide
hydrolysis in the function of dinitrogenase reductase has been compared
to the role of GTP hydrolysis in G proteins (8). MgATP
hydrolysis is coupled to the transfer of electrons from dinitrogenase
reductase to dinitrogenase. Seefeldt and coworkers have characterized
several altered forms of dinitrogenase reductase in which mutations
have been made in the nucleotide binding region (10, 11,
21). Substitution of Glu for Asp 129 resulted in a dinitrogenase
reductase that could still interact with dinitrogenase but that failed
to hydrolyze MgATP, and thus no electron transfer occurred
(11). Substitution of Gln for Asp 43 resulted in a
dinitrogenase reductase that had a decreased affinity for dinitrogenase
and that was unable to support substrate reduction (W. N. Lanzilotta and L. C. Seefeldt, unpublished data). Seefeldt and
coworkers have also characterized an altered dinitrogenase reductase in
which Leu 127 was deleted (Leu127) (10, 21). Leu 127 is
located between Asp 125, which is in the nucleotide binding site and
which interacts with the Mg2+ ion associated with the
nucleotide (23), and Cys 132, which is a ligand for the
Fe4S4 cluster (7).
, and D43N dinitrogenase reductases bound
MgADP (11, 21; Lanzilotta and Seefeldt, unpublished data); since
MgADP was added to the reactions, the dinitrogenase reductases studied
were in their MgADP-bound forms. D129E dinitrogenase reductase was
ADP-ribosylated when incubated with DRAT and NAD (Fig. 2, lane 6b).
Cross-linking studies showed that D129E dinitrogenase reductase
could also form a cross-linkable complex with DRAT (Fig. 3, lane
6). Furthermore, L127 dinitrogenase reductase could also be
ADP-ribosylated by DRAT (Fig. 2, lane 7b) and could form a cross-linkable complex with DRAT (Fig. 3, lane 7). In contrast, D43N was not a substrate for ADP-ribosylation (Fig. 2, lane 5b) and also could not form a cross-linkable complex with DRAT (Fig. 3,
lane 5). The major observable difference between these altered dinitrogenase reductases is that D43N dinitrogenase reductase has a
much weaker affinity for dinitrogenase (Lanzilotta and Seefeldt, unpublished data) than does either D129E (11) or L127
(21) dinitrogenase reductase. It is believed that DRAT and
dinitrogenase interact with similar regions of dinitrogenase reductase.
Therefore, the D43N mutation may cause a much greater overall
conformational change in dinitrogenase reductase, thereby affecting the
ADP-ribosylation site.
Nucleotide dependence of the ADP-ribosylation of L127
dinitrogenase reductase.
We have shown that L127
dinitrogenase reductase with MgADP bound is a substrate for
ADP-ribosylation and can form a cross-linkable complex with DRAT.
Seefeldt and coworkers (21) have shown that the
Fe4S4 cluster of L127 dinitrogenase
reductase has properties very similar to the
Fe4S4 cluster of wild-type dinitrogenase
reductase with MgATP bound, whether or not MgATP is bound to the
L127 protein. Their results suggest that deletion of Leu 127 brings
dinitrogenase reductase into an "MgATP-bound state" in the absence
of any bound nucleotides (21). However, the following
studies on the ADP-ribosylation properties of L127 dinitrogenase
reductase with various adenine nucleotides bound clearly showed that
there is a distinct difference between the nucleotide-free and
MgATP-bound forms of L127 dinitrogenase reductase as perceived by DRAT.
dinitrogenase reductase was incubated with DRAT, NAD, and
various nucleotides and analyzed for its ability to be ADP-ribosylated. L127 dinitrogenase reductase can be ADP-ribosylated in its
nucleotide-free form (Fig. 5, lane 1),
and in its MgADP-bound form (Fig. 5, lane 3). However, MgATP inhibits
the ADP-ribosylation of L127 dinitrogenase reductase by DRAT (Fig.
5, lane 6). To ensure that in this reaction all the L127
dinitrogenase reductase was in its MgATP-bound form, an ATP-generating
system (phosphocreatine and creatine phosphokinase) was added to this
reaction to convert any residual ADP to ATP. Control experiments showed
that this ATP-generating system had no effect on the ADP-ribosylation
of nucleotide-free L127 dinitrogenase reductase (Fig. 5, lane 4).
The following cross-linking studies between DRAT and L127
dinitrogenase reductase also showed that there is a difference in the
conformation of L127 dinitrogenase reductase in its nucleotide-free
form (Fig. 6B, lane 1) and in its
MgATP-bound form (Fig. 6B, lane 3). The extent of formation of a
cross-linkable complex between DRAT and L127 dinitrogenase reductase
is decreased when L127 dinitrogenase reductase is in its MgATP-bound
form versus when it is in its nucleotide-free form. This decrease in
cross-linking is also observed with DRAT and nucleotide-free or
MgATP-bound wild-type dinitrogenase reductase (Fig. 6A, lanes 1 to 3).
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dinitrogenase reductase
Fe4S4 cluster has very similar properties when
the protein is in its nucleotide-free or MgATP-bound form
(21), other regions of L127 dinitrogenase reductase,
particularly the region that interacts with DRAT, are clearly in a
different conformation depending on the nucleotide state of the protein.
Substitution of Gln for Lys 143 does not affect the binding of dinitrogenase reductase to DRAT. Seefeldt and coworkers have shown that Lys 143 of dinitrogenase reductase is important for the docking of dinitrogenase reductase with dinitrogenase (22). Changing Lys 143 to Gln resulted in a decreased affinity of the altered dinitrogenase reductase for binding to dinitrogenase (22). The crystal structure of dinitrogenase reductase shows Lys 143 positioned near the Arg 100 residue (4). To determine if the Lys 143 residue is also important for the interaction of dinitrogenase reductase with DRAT, K143Q dinitrogenase reductase was tested for its ability to be ADP-ribosylated and to be cross-linked to DRAT.
When K143Q dinitrogenase reductase was added to DRAT and NAD in an ADP-ribosylation reaction, 30% of the K143Q dinitrogenase reductase population was ADP-ribosylated (Fig. 2, lane 3b), compared to 78% of the wild-type dinitrogenase reductase population (Fig. 2, lane 1b). Cross-linking studies showed that K143Q dinitrogenase reductase and DRAT can interact and form a cross-linkable complex (Fig. 3, lane 3). Therefore, the substitution of Gln for Lys 143 has only slight effects on the interaction between DRAT and dinitrogenase reductase; therefore, dinitrogenase reductase Lys 143 is not a critical residue for the ADP-ribosylation of dinitrogenase reductase. In contrast, Lys 143 of dinitrogenase reductase is important in the interaction of dinitrogenase with dinitrogenase reductase. These data suggest that DRAT and dinitrogenase may interact with similar, but not identical, sites of dinitrogenase reductase. A number of NAD binding proteins contain in their active sites arginine residues which interact with NAD. The crystal structure of horse liver alcohol dehydrogenase shows two arginine residues in the NAD binding site (3). The side chain of Arg 47 is hydrogen bonded to the adenine-proximal phosphate group of NAD, and the side chain of Arg 369 is hydrogen bonded to the nicotinamide-proximal phosphate group of NAD. Li et al. have confirmed the role of these arginines in NAD binding by analyzing the crystal structure of alcohol dehydrogenase bound to an analog of NAD (thiazole-4-carboxamide adenine dinucleotide) (12). The crystal structure for Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ shows the side chain of Arg 266 hydrogen bonded to a water molecule which is hydrogen bonded to the nicotinamide ribose hydroxyl group (17). Furthermore, diptheria toxin (DT) bound to NAD has also been crystallized, and the NAD binding site has been characterized (1). His 21 is hydrogen bonded to the adenosine ribose hydroxyl group of NAD. Bell and Eisenberg (1) structurally aligned the NAD binding sites of Escherichia coli heat-labile enterotoxin and pertussis toxin to the NAD binding site of DT. They showed that Arg 7 of heat-labile enterotoxin and Arg 9 of pertussis toxin are in the same position in the NAD binding pocket as is His 21 of DT. Therefore, these arginine residues may play a role similar to that of His 21 in binding to the adenine-proximal ribose ring or possibly to the adenine-proximal phosphate group of NAD. The sites of binding of NAD to ADP-ribosyltransferases appear to be structural motifs distinctly different from the well-characterized Rossman fold observed for the dehydrogenases (2). The N-C bond between the nicotinamide and ribose groups of NAD must be oriented near Arg 100 of dinitrogenase reductase for ADP-ribosylation of Arg 100 to occur. With this as a base position for NAD, molecular modeling (performed by Wayne Schultz) of the NAD molecule in its DT-bound conformation (1) allows us to speculate about the interaction of NAD and A. vinelandii dinitrogenase reductase. This molecular modeling shows that the NAD molecule could interact with both Arg 100 and Arg 140. Two possible orientations for NAD on dinitrogenase reductase are that (i) the NAD molecule is positioned such that it bridges the two subunits of dinitrogenase reductase or (ii) it is positioned in the cleft of the two subunits of dinitrogenase reductase. The crystal structure of dinitrogenase reductase reported by Georgiadis et al. (4) shows that the distance between the guandino nitrogens of the Arg 100 and Arg 140 side chains located in the same subunit is approximately 11 Å and the distance between the guandino nitrogens of the Arg 100 and Arg 140 side chains located in different subunits is approximately 8 Å (4). For the NAD molecule bound to DT, the distance between the nitrogen atom of the nicotinamide ring and the adenine-proximal phosphate group is approximately 7 Å (1). Arg 140 of dinitrogenase reductase is required for the ADP-ribosylation of dinitrogenase reductase. By analogy to the previously described NAD binding proteins, Arg 140 may be involved in hydrogen bonding to either the ribose hydroxyl groups or the phosphate groups of NAD during DRAT-catalyzed ADP-ribosylation.Conclusions.
The following conclusions can be made about the
region of dinitrogenase reductase that is important for the
ADP-ribosylation of dinitrogenase reductase. (i) The amino acid Arg 140 is required for dinitrogenase reductase to be ADP-ribosylated. The side
chain of Arg 140 may hydrogen bond to the phosphate or ribose groups of
NAD and thus help position NAD in the proper conformation for ADP-ribosylation of dinitrogenase reductase to occur. (ii) A native conformation in the Fe4S4 region of
dinitrogenase reductase is also required for ADP-ribosylation to occur,
as evidenced by the inability of F135Y and apo-dinitrogenase reductase
to be ADP-ribosylated. (iii) The conformation of the L127
dinitrogenase reductase is readily recognized by DRAT; complex
formation with DRAT and ADP-ribosylation of L127 dinitrogenase
reductase are inhibited by MgATP.
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ACKNOWLEDGMENTS |
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We thank Gary P. Roberts, Lance Seefeldt, and James Howard for advice and useful suggestions.
This work was supported by NIH grant GM54910 to P.W.L. S.K.G. was supported by NIH training grant 5T32 GM07215 during a portion of this study.
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FOOTNOTES |
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*
Corresponding author. Mailing address: Department of
Biochemistry and Center for the Study of Nitrogen Fixation, College of Agricultural and Life Sciences, University of WisconsinMadison, 433 Babcock Dr., Madison, WI 53706-1544. Phone: (608) 262-6859. Fax: (608)
262-3453. E-mail: ludden{at}biochem.wisc.edu.
Present address: Department of Chemistry, University of
WisconsinLa Crosse, La Crosse, WI 54601.
Present address: Biochemistry Department, Michigan State
University, East Lansing, MI 48824.
§
Present address: Department of Molecular Biology and Biochemistry,
University of CaliforniaIrvine, Irvine, CA 92697.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Journal of Bacteriology, May 2000, p. 2597-2603, Vol. 182, No. 9
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This article has been cited by other articles:
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