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1. NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?

   https://doi.org/10.1128/AEM.01758-20  

   O’Neill, Audrey G.; Beaupre, Brett A.; Zheng, Yuanzhang; Liu, Dali; Moran, Graham R. (October 2020, Applied and Environmental Microbiology)

   Pettinari, M. Julia (Ed.)

   ABSTRACT Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus , which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y ). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s −1 . Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s −1 . The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes. IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed. 

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2. Not Once, not Twice, but Thrice: Structure and Catalytic Mechanisms of the Dihydroorotate Dehydrogenases

   https://doi.org/10.1021/acs.biochem.5c00170  

   Smith, Corine O; Moran, Graham R (June 2025, Biochemistry)

   Dihydroorotate dehydrogenases (DHODs) are common to all life and catalyze the oxidation of dihydroorotate (DHO) to orotate the precursor of all pyrimidine nucleotides. The core structure of all DHODs has a TIM-barrel topology (the PyrD subunit or domain) that harbors an FMN cofactor that interacts with DHO. There are two classes of DHOD enzymes. Each has unique structures and oxidant substrates that conserve part of the energy available by coupling the reaction to ATP synthesis. The class 1 enzymes are soluble and divided into classes 1A and 1B. Class 1A has fumarate as the electron acceptor forming succinate and is the simplest form of DHOD, successively binding DHO and fumarate at the same active site locale. Class 1B uses NAD+ as the oxidant and this form of DHOD is heterodimeric having, in addition to the PyrD subunit, a subunit (PyrK) whose structure is like those of ferredoxin reductases. PyrK adds a second active site with a bound FAD that interacts with the NAD+ substrate and includes an Fe2S2 center that resides at the interface of the subunits, forming a conduit for electrons. Class 2 DHODs have ubiquinone (UQ) as the electron acceptor. This form of DHOD is membrane associated via an N-terminal domain that also forms a quinone binding site end-on to the FMN xylene moiety. This arrangement uses the flavin to mediate between the substrates and as a redox partition between water-soluble NAD+ and lipid soluble UQ10. In this review, we summarize the structure and mechanism of DHOD enzymes. 

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3. Solvation stabilizes intercarbonyl n→π* interactions and polyproline II helix

   https://doi.org/10.1039/D2CP00857B  

   Zondlo, Neal J. (June 2022, Physical Chemistry Chemical Physics)

   n→π* interactions between consecutive carbonyls stabilize the α-helix and polyproline II helix (PPII) conformations in proteins. n→π* interactions have been suggested to provide significant conformational biases to the disordered states of proteins. To understand the roles of solvation on the strength of n→π* interactions, computational investigations were conducted on a model n→π* interaction, the twisted-parallel-offset formaldehyde dimer, as a function of explicit solvation of the donor and acceptor carbonyls, using water and HF. In addition, the effects of urea, thiourea, guanidinium, and monovalent cations on n→π* interaction strength were examined. Solvation of the acceptor carbonyl significantly strengthens the n→π* interaction, while solvation of the donor carbonyl only modestly weakens the n→π* interaction. The n→π* interaction strength was maximized with two solvent molecules on the acceptor carbonyl. Urea stabilized the n→π* interaction via simultaneous engagement of both oxygen lone pairs on the acceptor carbonyl. Solvent effects were further investigated in the model peptides Ac-Pro-NMe 2 , Ac-Ala-NMe 2 , and Ac-Pro 2 -NMe 2 . Solvent effects in peptides were similar to those in the formaldehyde dimer, with solvation of the acceptor carbonyl increasing n→π* interaction strength and resulting in more compact conformations, in both the proline endo and exo ring puckers, as well as a reduction in the energy difference between these ring puckers. Carbonyl solvation leads to an energetic preference for PPII over both the α-helix and β/extended conformations, consistent with experimental data that protic solvents and protein denaturants both promote PPII. Solvation of the acceptor carbonyl weakens the intraresidue C5 hydrogen bond that stabilizes the β conformation. 

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4. TrmB Family Transcription Factor as a Thiol-Based Regulator of Oxidative Stress Response

   https://doi.org/10.1128/mbio.00633-22  

   Mondragon, Paula; Hwang, Sungmin; Kasirajan, Lakshmi; Oyetoro, Rebecca; Nasthas, Angelina; Winters, Emily; Couto-Rodriguez, Ricardo L.; Schmid, Amy; Maupin-Furlow, Julie A. (August 2022, mBio)

   Babitzke, Paul (Ed.)

   ABSTRACT Oxidative stress causes cellular damage, including DNA mutations, protein dysfunction, and loss of membrane integrity. Here, we discovered that a TrmB (transcription regulator of mal operon) family protein (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulator of oxidative stress response. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons, including genes associated with thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel α helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea. IMPORTANCE TrmB-like proteins, while not yet associated with redox stress, are found in bacteria and widespread in archaea. Here, we expand annotation of a large group of TrmB-like single winged-helix DNA binding domain proteins from diverse archaea to function as thiol-based transcriptional regulators of oxidative stress response. Using Haloferax volcanii as a model, we reveal that the TrmB-like OxsR functions during hypochlorite stress as a transcriptional activator and repressor of an extensive gene coexpression network associated with thiol relay and other related activities. A conserved cysteine residue of OxsR serves as the thiol-based sensor for this function and likely forms an intersubunit disulfide bond during hypochlorite stress that stabilizes a homodimeric configuration with enhanced DNA binding properties. A CG-rich DNA motif in the promoter region of a subset of sites identified to be OxsR-bound is required for regulation; however, not all sites have this motif, suggesting added complexity to the regulatory network. 

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5. Biochemical and structural characterization of an aromatic ring–hydroxylating dioxygenase for terephthalic acid catabolism

   https://doi.org/10.1073/pnas.2121426119  

   Kincannon, William M.; Zahn, Michael; Clare, Rita; Lusty Beech, Jessica; Romberg, Ari; Larson, James; Bothner, Brian; Beckham, Gregg T.; McGeehan, John E.; DuBois, Jennifer L. (March 2022, Proceedings of the National Academy of Sciences)

   Several bacteria possess components of catabolic pathways for the synthetic polyester poly(ethylene terephthalate) (PET). These proceed by hydrolyzing the ester linkages of the polymer to its monomers, ethylene glycol and terephthalate (TPA), which are further converted into common metabolites. These pathways are crucial for genetically engineering microbes for PET upcycling, prompting interest in their fundamental biochemical and structural elucidation. Terephthalate dioxygenase (TPADO) and its cognate reductase make up a complex multimetalloenzyme system that dihydroxylates TPA, activating it for enzymatic decarboxylation to yield protocatechuic acid (PCA). Here, we report structural, biochemical, and bioinformatic analyses of TPADO. Together, these data illustrate the remarkable adaptation of TPADO to the TPA dianion as its preferred substrate, with small, protonatable ring 2-carbon substituents being among the few permitted substrate modifications. TPADO is a Rieske [2Fe2S] and mononuclear nonheme iron-dependent oxygenase (Rieske oxygenase) that shares low sequence similarity with most structurally characterized members of its family. Structural data show an α-helix–associated histidine side chain that rotates into an Fe (II)–coordinating position following binding of the substrate into an adjacent pocket. TPA interactions with side chains in this pocket were not conserved in homologs with different substrate preferences. The binding mode of the less symmetric 2-hydroxy-TPA substrate, the observation that PCA is its oxygenation product, and the close relationship of the TPADO α-subunit to that of anthranilate dioxygenase allowed us to propose a structure-based model for product formation. Future efforts to identify, evolve, or engineer TPADO variants with desirable properties will be enabled by the results described here. 

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