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Enzyme Structure and Function


Previous studies on bacterial acetone metabolism both in vivo and in vitro suggested that acetone can be metabolized in two ways: an oxygen-dependent monooxygenase-catalyzed oxidation to produce acetol (hydroxyacetone), or a CO2-dependent carboxylase-catalyzed carboxylation to produce acetoacetate. The final carboxylation step of both propylene and acetone metabolism demonstrates the use of high energy intermediates to overcome the activation energy of bond formation. These intermediates create enolacetone as a strong nucleophile and encapsulate CO2 to prevent side reactions. The Peters’ lab has done structural studies of both 2-KPCC and AC have demonstrated how enolacetone forms and is protected from solvent exposure.

Acetone carboxylase

Acetone carboxylases (ACs) catalyze the conversion of acetone and HCO3 to form acetoacetate. ACs contain metal ions and use ATP to activate substrates through phosphorylation. The identity of the metal, however, has been under scrutiny. Analysis of AC from Xanthobacter autotrophicus and Rhobacter capsulatus identified Mn at the active site while Fe was identified in Aromatoleum aromaticum. The stoichiometry of acetone consumption:ATP hydrolysis was also reported to be 1:1 in X. autorophicus and 1:2 in A. aromaticum. The (abg)2 heterohexameric structure for X. autorophicus AC was shown to undergo large conformational changes in the presence of AMP and/or acetate. It has been proposed that the rotation encapsulates the substrates ATP, HCO3, and acetone at the nucleotide binding site where the substrates are phosphorylated (phosphoenolacetone and carboxyphosphate). During this rotation, a glutamate residue (E89) coordinates the metal site, facilitating an open channel from the nucleotide site to the metal site. The phosphorylated intermediates are shuttled from the nucleotide binding site to the metal site where, presumably, they displace E89 and coordinate the metal. Following dephosphorylation, the enolacetone attacks CO2, forming acetoacetate. The Peters’ lab is currently investigating the identity of the metal center and ATP stoichiometry in ACs from different species, structural changes throughout catalysis, in addition to mechanistic details.


The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate in a controlled reaction to exclude protons. 2-KPCC is the only member of the disulfide oxidoreductase (DSOR) family of enzymes to have carboxylation activity. 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes that facilitate catalysis. These conformational changes promote substrate discrimination between H+ and CO2 to favor the carboxylation product, acetoacetate. The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to typical DSORs in which the active-site histidine had been replaced with an
alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. A single proton is shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C–S bond of the substrate. A conserved His–Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe–His. This difference is important for coupling carboxylation with C–S bond cleavage. The Phe–His dyad in 2-KPCC has been substituted to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E). F501 was shown to protect the enolacetone intermediate from protons while the H506 residue stabilizes the developing charge during the formation of acetoacetate. The F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Additionally, the active site dyad is important for transducing a ligand induced conformational change that excludes H+ and facilitates the binding of CO2 to promote carboxylation. The 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.


Biological Nitrogen Fixation


Access to fixed or available forms of N often limits the productivity of crop plants and thus the production of food, feed, fiber, and fuel. Since the Green
Revolution, N fertilizers provided by the Haber–Bosch process have become an essential part of modern agriculture, sustaining crop yields and replacing N removed from the system at harvest. However, with the increasing global population, problems caused by unintended N leaching and the production of greenhouse gases have led to a global “nitrogen problem”. More sustainable ways of managing the N cycle in soil and utilizing biological nitrogen fixation (BNF) are now imperative. BNF is a process that select microbes use to convert nitrogen gas to ammonia that can be utilized by plants and also makes a contribution to agricultural productivity. There is growing interest in engineered soil diazotrophs producing ammonium in excess that promote the growth of plants under fixed nitrogen-limiting conditions, and can potentially provide a means to reduce the use of synthetic nitrogen fertilizers, thus providing a solution to the “nitrogen problem”.

In the ubiquitous diazotrophic soil bacterium Azotobacter vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an anti-activator that tightly controls the activity of the nitrogen fixation specific transcriptional activator, NifA, in response to redox, nitrogen, and carbon status. The mis-regulation of BNF in A. vinelandii, through mutations of the key regulator nifL, can lead to ammonia excretion. However knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. Our research is focusing on the fundamental understanding of the mechanisms involved in the regulation of BNF and examining the discrete genetic determinants associated with ammonia excretion. The outcome of our research will provide important new insights for developing a blueprint for engineering microorganisms for effective biofertilizers.

Access to available nitrogen commonly limits plant growth and the agricultural demand for nitrogen is largely supplemented with synthetic nitrogen fertilizers, which cause deleterious environmental effects including soil acidification and greenhouse gas emissions . Diazotrophs are bacteria and archaea that use the enzyme nitrogenase to reduce atmospheric nitrogen into ammonia that plants readily incorporate into biomolecules. Using diazotrophs in agriculture provides an avenue to reduce dependence on synthetic fertilizers, thereby improving land stewardship and agricultural sustainability. The goal of our research is to understand how diazotrophs regulate nitrogenase, knowledge that is required to engineer high-performance biofertilizers from diazotrophs compatible with a wide variety of crops. We use the model diazotroph Azotobacter vinelandii, in which nitrogenase expression is controlled by the transcriptional activator NifA and its repressor NifL, to investigate the physiological roles of NifL domains in nitrogenase regulation and elucidate the mechanism by which signal-induced conformational changes in NifL modulate its interaction with NifA. Successful completion of the project aims will advance scientific understanding of the mechanism of NifL-NifA nitrogenase regulation, facilitating engineering efforts to develop associative diazotrophic biofertilizers that are compatible with modern agriculture.

Catalytic Bias

Clostridium pasteurianum [FeFe]-hydrogenases

Catalytic bias refers to the relative rate preference of a catalyst for either the forward or reverse direction. In metal cofactor-based redox catalysis, the tuning of catalytic bias plays an underlying role in controlling rates of reactivity. For this, enzymes have evolved complex active sites that can exist in multiple oxidation states with differing reduction potentials in order to achieve challenging multi-step, redox reactions. Conceivably, the relative stability of the intermediates that contribute to determining the rate-limiting step of the catalytic cycle could impose catalytic bias, although mechanisms for this concept are just beginning to be understood. As one example, recent work on Clostridium pasteurianum [FeFe]-hydrogenases, which catalyze reversible hydrogen oxidation have shown that the differential stabilization/destabilization of active site oxidation states through static and/or dynamic protein interactions can preferentially promote either the hydrogen oxidation or proton reduction direction of the reaction. This revealed how an enzymatic cofactor can impose bias in redox catalysis through various tuning mechanisms by protein scaffold interact­ions. The hypothesis based on achieving catalytic bias through the modulation of cofactor oxidation states critical for the reaction cycle can be extended more generally to other cofactor-based oxidation-reductions catalysts. The current understanding of catalytic bias would have significant implications towards the design of novel synthetic catalysts used in industrial and commercial settings, as well as providing a greater fundamental understanding into the factors that control metabolic processes in all life.



Electron bifurcation (EB) was first described by Peter Mitchell 40 years ago to explain how complex III of the aerobic respiratory chain coupled quinol (QH2) oxidation in the mitochondrial inner membrane to quinone (Q) reduction in the matrix via the Q-cycle. EB was thought to be a unique feature of complex III, until a decade ago it was discovered that flavin-based electron bifurcation (FBEB) is an important component of microbial anaerobic metabolism. In FBEB, the flavin site allows the oxidation of NAD(P)H and bifurcates the electron pair to reduce the low-potential redox protein ferredoxin or flavodoxin (endergonic reaction) by using the free energy released by the reduction of unsaturated organics, quinones, or disulfides. The reduced ferredoxin or flavodoxin is used as an electron donor in anaerobic metabolism to drive important metabolic reactions.

The obligate aerobe A. vinelandii contains a membrane-bound complex called FixABCX encoded by six genes: fixFd, fixA, fixB, fixC, fix, and ORF6, where fixFd (encoding a small iron-sulfur protein) and ORF6 (encoding a ferritin-like protein) are only identified in the fix operon of A. vinelandii. The FixABCX complex has been biochemically characterized to show a new pathway for the generation of low-potential reductant ferredoxin/flavodoxin needed for the energetically-demanding reduction of dinitrogen to ammonia by nitrogenase. As the FixABCX complex is a member of the ETF family of enzymes, a conformational gating of electron transfer is proposed to occur during its catalytic cycle. To define the mechanism of conformational gating, we are using cryoEM to understand the structure and site-specific labelling and HDX MS to examine the protein conformations in the oxidized and reduced states.