What They Knew
Nitrogen fixation is a demanding process, using a lot of energy, so bacteria regulate it tightly, shutting it off whenever fixed nitrogen is available already. One diazotroph, Rhodospirillum rubrum, regulates its nitrogenase by ADP-ribosylating the dinitrogenase reductase component, using a protein called Dinitrogenase Reductase ADP-ribosyl Transferase, or DRAT. This takes ADP-ribose from NAD when ammonium is present. Another protein, Dinitrogenase Reductase-Activating Glycohydrolase (DRAG), reverses the process.
What They Wanted to Know
Ponnuraj and colleagues studied the nitrogenases of R. rubrum and Azotobacter vinelandii to see how specifically each interacted with the DRAT/DRAG system and various ADP-ribosyl donors.
What They Did
They took different ADP-ribosyl containing molecules (NAD, NADP, NADH, NMN), combined each with A. vinelandii's dinitrogenase reductase (DNR) and R. rubrum's DRAT, then ran them with SDS-PAGE along with samples lacking the molecules, to see which molecules could be used to donate ADP-ribosyl. They also exposed some of each sample to DRAG and ran that alongside to see if it could remove the modification.
Results from this were confirmed with another test, seeing if modified or de-modified DNR could function with dinitrogenase to reduce acetylene.
To see how small a modification works to inactivate the system, they removed a phosphate from phosphoribosylated DNR and tested it again.
In addition to testing A. vinelandii's DNR, they tested R. rubrum's too.
To see more specifically what was going on with A. vinelandii's DNR, they used MALDI-TOF mass spectroscopy.
What They Observed
Based on gels and activity assays, NAD, NMN, and NADP all seemed pretty good at donating to DRAT to inactivate A. vinelandii's DNR. NAAD not so much, or anything else they tried. DRAG seemed able to re-activate DNR with all of them too.
Surprisingly, with R. rubrum's DNR, only NAD seemed to be a good donor. Mass spec results confirmed their expectations about what was going on biochemically.
What This Means
R. rubrum uses DRAT and DRAG to regulate its nitrogenase activity based on whether fixed nitrogen is available already and whether its environment is illuminated or not. This helps save energy, so it doesn't have to break down the whole system and reconstruct it with every little environmental change.
It's not clear how relevant it is for A. vinelandii, though, because that organism doesn't appear to have the genes to produce DRAT/DRAG proteins. It's somewhat interesting that R. rubrum's proteins are able to modify A. vinelandii's nitrogenase, arguably even better than they can with R. rubrum's, but this actually makes some sense, that R. rubrum would have tighter control over its nitrogenase regulation system. Though apparently some in vivo studies suggest it might not be as tight as it seemed here.
This doesn't necessarily mean that A. vinelandii doesn't have a system for post-translational regulation, just that it isn't exactly this one. I haven't found what it is yet, if there is one. And other studies seem to imply that A. vinelandii might not have such tight control (010). I wonder why.
Nitrogen fixation is a demanding process, using a lot of energy, so bacteria regulate it tightly, shutting it off whenever fixed nitrogen is available already. One diazotroph, Rhodospirillum rubrum, regulates its nitrogenase by ADP-ribosylating the dinitrogenase reductase component, using a protein called Dinitrogenase Reductase ADP-ribosyl Transferase, or DRAT. This takes ADP-ribose from NAD when ammonium is present. Another protein, Dinitrogenase Reductase-Activating Glycohydrolase (DRAG), reverses the process.
What They Wanted to Know
Ponnuraj and colleagues studied the nitrogenases of R. rubrum and Azotobacter vinelandii to see how specifically each interacted with the DRAT/DRAG system and various ADP-ribosyl donors.
What They Did
They took different ADP-ribosyl containing molecules (NAD, NADP, NADH, NMN), combined each with A. vinelandii's dinitrogenase reductase (DNR) and R. rubrum's DRAT, then ran them with SDS-PAGE along with samples lacking the molecules, to see which molecules could be used to donate ADP-ribosyl. They also exposed some of each sample to DRAG and ran that alongside to see if it could remove the modification.
Results from this were confirmed with another test, seeing if modified or de-modified DNR could function with dinitrogenase to reduce acetylene.
To see how small a modification works to inactivate the system, they removed a phosphate from phosphoribosylated DNR and tested it again.
In addition to testing A. vinelandii's DNR, they tested R. rubrum's too.
To see more specifically what was going on with A. vinelandii's DNR, they used MALDI-TOF mass spectroscopy.
What They Observed
Based on gels and activity assays, NAD, NMN, and NADP all seemed pretty good at donating to DRAT to inactivate A. vinelandii's DNR. NAAD not so much, or anything else they tried. DRAG seemed able to re-activate DNR with all of them too.
Surprisingly, with R. rubrum's DNR, only NAD seemed to be a good donor. Mass spec results confirmed their expectations about what was going on biochemically.
What This Means
R. rubrum uses DRAT and DRAG to regulate its nitrogenase activity based on whether fixed nitrogen is available already and whether its environment is illuminated or not. This helps save energy, so it doesn't have to break down the whole system and reconstruct it with every little environmental change.
It's not clear how relevant it is for A. vinelandii, though, because that organism doesn't appear to have the genes to produce DRAT/DRAG proteins. It's somewhat interesting that R. rubrum's proteins are able to modify A. vinelandii's nitrogenase, arguably even better than they can with R. rubrum's, but this actually makes some sense, that R. rubrum would have tighter control over its nitrogenase regulation system. Though apparently some in vivo studies suggest it might not be as tight as it seemed here.
This doesn't necessarily mean that A. vinelandii doesn't have a system for post-translational regulation, just that it isn't exactly this one. I haven't found what it is yet, if there is one. And other studies seem to imply that A. vinelandii might not have such tight control (010). I wonder why.
Reference:
Ponnuraj, R. K., Rubio, L. M., Grunwald, S. K. & Ludden, P. W. NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii. FEBS Letters 579, 5751–5758 (2005).
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