A doubly substituted form of the nitrogenase MoFe protein (α-70 Val→Ala , α-195 His→Gln ) has the capacity to catalyze the reduction of carbon dioxide (CO 2 ) to yield methane (CH 4 ). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH 4 within 20 min. The catalytic rate depends on the partial pressure of CO 2 (or concentration of HCO 3 − ) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H 2 C = CH-CH 3 ) through the reductive coupling of CO 2 and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO 2 sequestration and formation of olefins.is an abundant and stable form of carbon that is the product of respiration and burning of fossil fuels. As a result of these activities, the atmospheric concentration of CO 2 , a greenhouse gas, has been rising over the last century and contributing to global warming (1). There is strong interest in developing methods for sequestering CO 2 either by capturing it or by chemically converting it to valuable chemicals (2-5). Of particular interest are possible routes to reduction of CO 2 by multiple electrons to yield methanol (CH 3 OH) and methane (CH 4 ), which are renewable fuels (2). The reduction of CO 2 is difficult, with a limited number of reports of metal-based compounds able to catalyze these reactions (6-13). In biology, only a few enzymes are known to reduce CO 2 (14-18), and none of these can catalyze the eight electron reduction to CH 4 .The bacterial Mo-dependent nitrogenase enzyme catalyzes the multielectron/proton reduction of dinitrogen (N 2 ) to two ammonia (NH 3 ) at a metal cluster designated FeMo-cofactor [7Fe-9S-1Mo-1C-R-homocitrate] (Fig. 1) in a reaction that requires ATP hydrolysis and evolution of H 2 , with a minimal reaction stoichiometry shown in Eq. 1 (19)(20)(21)(22).Given that nitrogenase is effective at catalyzing the difficult multielectron reduction of N 2 , it was of interest to determine whether this enzyme might also catalyze the reduction of CO 2 to the level of CH 4 . Nitrogenase is known to have the capacity to reduce a variety of other small, relatively inert, doubly or triply bonded compounds, such as acetylene (HC≡CH) (19,23). It has been shown that an alternative form of nitrogenase, which contains V in place of Mo in the active site cofactor, has the remarkable capacity to reduce CO and couple multiple CO molecules, yielding short chain alkenes and alkanes such as ethylene (C 2 H 4 ), ethane (C 2 H 6 ), propylene (C 3 H 6 ), and propane (C 3 H 8 ) (24,25). In contrast, the Mo-nitrogenase is only able to reduce CO at exceedingly low rates (24). However, we have found that the MoFe protein can be remodeled by substitution of amino acid residues that provide the first shell of noncovalent interactions with the active site FeMo cofa...

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The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that P(II) proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used (1)H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. P(II) proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P(II) by introducing a thermal treatment step in the P(II) purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR(1) and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P(II) protein family members.

NADH (NAD) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadE and octameric glutamine-dependent NadE, and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadE in bacteria. Substrate preferences and structural analyses suggested that dimeric NadE enzymes may constitute evolutionary intermediates between dimeric NadE and octameric NadE The characterization of additional NadE isoforms in the diazotrophic bacterium along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes.

Posttranslational modification of proteins plays a key role in the regulation of a plethora of metabolic functions. Protein modification by mono-ADP-ribosylation was first described as a mechanism of action of bacterial toxins. Since these pioneering studies, the number of pathways regulated by ADP-ribosylation in organisms from all domains of life expanded significantly. However, in only a few cases the full regulatory ADP-ribosylation circuit is known. Here, we review the system where mono-ADP-ribosylation regulates the activity of an enzyme: the regulation of nitrogenase in bacteria. When the nitrogenase product, ammonium, becomes available, the ADP-ribosyltransferase (DraT) covalently links an ADP-ribose moiety to a specific arginine residue on nitrogenase switching-off nitrogenase activity. After ammonium exhaustion, the ADP-ribosylhydrolase (DraG) removes the modifying group, restoring nitrogenase activity. DraT and DraG activities are reversibly regulated through interaction with PII signaling proteins . Bioinformatics analysis showed that DraT homologs are restricted to a few nitrogen-fixing bacteria while DraG homologs are widespread in Nature. Structural comparisons indicated that bacterial DraG is closely related to Archaea and mammalian ADP-ribosylhydrolases (ARH). In all available structures, the ARH active site consists of a hydrophilic cleft carrying a binuclear Mg(2+) or Mn(2+) cluster, which is critical for catalysis.

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Plant growth stimulation by microorganisms that interact in a mutually beneficial manner remains poorly understood. Understanding the nature of plant-bacteria interactions may open new routes for plant productivity enhancement, especially cereal crops consumed by humans. Proteomic and metabolomic analyses are particularly useful for elucidating these mechanisms. A complete depiction of these mechanisms will prompt researchers to develop more efficient plant-bacteria associations. The success of microorganisms as biofertilizers may replace the current massive use of chemical fertilizers, mitigating many environmental and economic issues. In this review, we discuss the recent advances and current state of the art in proteomics and metabolomics studies involving grass-bacteria associations. We also discuss essential subjects involved in the bacterial plant-growth promotion, such, nitrogen fixation, plant stress, defense responses, and siderophore production.

bFe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy in Azospirillum brasilense, Rhodospirillum rubrum, and Rhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling P II proteins. In A. brasilense, two P II proteins, GlnB and GlnZ, were identified. We used Fe protein from Azotobacter vinelandii as the substrate to assess the activity of A. brasilense DraT in vitro complexed or not with P II proteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The P II effector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated P II proteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that the A. brasilense DraT-GlnB complex was at least 18-fold more efficient than DraT purified from R. rubrum, but with a similar K m value for NAD ؉ . Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.