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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis

The flavin transferase ApbE plays essential roles in bacterial physiology, covalently incorporating FMN cofactors into numerous respiratory enzymes that use the integrated cofactors as electron carriers. In this work we performed a detailed kinetic and structural characterization of Vibrio cholerae WT ApbE and mutants of the conserved residue His-257, to understand its role in substrate binding and in the catalytic mechanism of this family. Bi-substrate kinetic experiments revealed that ApbE follows a random Bi Bi sequential kinetic mechanism, in which a ternary complex is formed, indicating that both substrates must be bound to the enzyme for the reaction to proceed. Steady-state kinetic analyses show that the turnover rates of His-257 mutants are significantly smaller than those of WT ApbE, and have increased K m values for both substrates, indicating that the His-257 residue plays important roles in catalysis and in enzyme-substrate complex formation. Analyses of the pH dependence of ApbE activity indicate that the p K a of the catalytic residue (p K ES1 ) increases by 2 pH units in the His-257 mutants, suggesting that this residue plays a role in substrate deprotonation. The crystal structures of WT ApbE and an H257G mutant were determined at 1.61 and 1.92 Å resolutions, revealing that His-257 is located in the catalytic site and that the substitution does not produce major conformational changes. In this paper, we propose a reaction mechanism in which His-257 acts as a general base that deprotonates the acceptor residue, which subsequently performs a nucleophilic attack on FAD for flavin transfer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Crystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2

Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X -Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

How Aqueous Solvation Impacts the Frequencies and Intensities of Infrared Absorption Bands in Flavin: The Quest for a Suitable Solvent Model

FTIR spectroscopy accompanied by quantum chemical simulations can reveal important information about molecular structure and intermolecular interactions in the condensed phase. Simulations typically account for the solvent either through cluster quantum mechanical (QM) models, polarizable continuum models (PCM), or hybrid quantum mechanical/molecular mechanical (QM/MM) models. Recently, we studied the effect of aqueous solvent interactions on the vibrational frequencies of lumiflavin, a minimal flavin model, using cluster QM and PCM models. Those models successfully reproduced the relative frequencies of four prominent stretching modes of flavin’s isoalloxazine ring in the diagnostic 1450–1750 cm−1 range but poorly reproduced the relative band intensities. Here, we extend our studies on this system and account for solvation through a series of increasingly sophisticated models. Only by combining elements of QM clusters, QM/MM, and PCM approaches do we obtain an improved agreement with the experiment. The study sheds light more generally on factors that can impact the computed frequencies and intensities of IR bands in solution.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Structural Basis of Sequential Enantioselective Epoxidation by a Flavin‐Dependent Monooxygenase in Lasalocid A Biosynthesis

Abstract Polyether polyketides are a structurally diverse group of natural products known for their antimicrobial and antiproliferative activities. Lasalocid A is a canonical natural polyether produced by the soil bacteriumStreptomyces lasalocidi. In lasalocid A biosynthesis, a polyene polyketide intermediate is converted into a bisepoxide by the flavin‐dependent monooxygenase enzyme Lsd18. Remarkably, Lsd18 acts on two distinct C═C groups in the substrate molecule, forming two (R,R) epoxides. We have determined the X‐ray crystal structures of Lsd18 in the substrate‐free, substrate‐bound, and product‐bound forms. Our work has revealed that Lsd18 has an extra‐large substrate‐binding pocket that allows the polyene to adopt different conformations within the enzyme pocket. This feature enables Lsd18 to epoxidate both of the C═C groups. Additionally, a subpocket located near the Lsd18 active site controls stereoselectivity by dictating which face of the C═C group is placed next to the flavin. Molecular understanding of how Lsd18 transforms a polyene into a bisepoxide during lasalocid A biosynthesis lays the foundation for the production of designer polyethers for drug development.

Chemistry↗
DOE OSTI2023

Triepoxide formation by a flavin-dependent monooxygenase in monensin biosynthesis

Monensin A is a prototypical natural polyether polyketide antibiotic. It acts by binding a metal cation and facilitating its transport across the cell membrane. Biosynthesis of monensin A involves construction of a polyene polyketide backbone, subsequent epoxidation of the alkenes, and, lastly, formation of cyclic ethers via epoxide-opening cyclization. MonCI, a flavin-dependent monooxygenase, is thought to transform all three alkenes in the intermediate polyketide premonensin A into epoxides. Our crystallographic study has revealed that MonCI’s exquisite stereocontrol is due to the preorganization of the active site residues which allows only one specific face of the alkene to approach the reactive C(4a)-hydroperoxyflavin moiety. Furthermore, MonCI has an unusually large substrate-binding cavity that can accommodate premonensin A in an extended or folded conformation which allows any of the three alkenes to be placed next to C(4a)-hydroperoxyflavin. Importantly, MonCI, with its ability to perform multiple epoxidations on the same substrate in a stereospecific manner, demonstrates the extraordinary versatility of the flavin-dependent monooxygenase family of enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

A bacterial flavin‐dependent oxidoreductase that captures carbon dioxide into biomass

Atmospheric carbon dioxide is used as a carbon source for building biomass in plants and photosynthetic microbes. Non‐photosynthetic processes that also fix carbon dioxide have more recently been discovered. This research focuses on a microbial mechanism for coupling acetone to CO 2 to make a central metabolite, acetoacetate. The key reaction is catalyzed by NADPH‐2‐ketopropyl‐coenzyme M oxidoreductase/carboxylase (2‐KPCC), a bacterial enzyme that is part of the flavin and cysteine‐disulfide containing oxidoreductase family (DSORs) which are best known for reducing metallic or disulfide substrates. Our research asks: how has nature repurposed a DSOR to, uniquely, break a C‐S bond and then trap and fix CO 2 ? 2‐KPCC lacks a conserved, catalytically essential acid‐base histidine possessed by all other DSOR enzymes, having instead a phenylalanine (F501) at the same position. Mutagenesis showed that a F501H mutant has a similar rate of catalytic turnover; however, the product is acetone instead of acetoacetate (Figure 1). We hypothesized that F501 is important for both the reductive half reaction – which generates the reactive, C‐S bond breaking form of the active site – and for the oxidative half, in which an enolacetone intermediate reacts with either the correct (CO 2 ) or incorrect (H + ) electrophile. In this study, we used real‐time and spectroscopic methods to examine the reductive half reaction. In typical DSORs, this reaction generates a Cys/FAD charge transfer species. However, we showed that 2‐KPCC generates an electronically unique form of the active site, in which the flavin is oxidized and a pair of active site histidines are reduced and protonated. We hypothesize that this form of the active site generates the substrate‐reactive Cys in a more nucleophilic form, where it is capable of cleaving relatively strong C‐S bonds. The resulting enolacetone carbanion is an extremely potent nucleophile that is capable of directly attacking CO 2 . Research on 2‐KPCC and other biological CO 2 fixation methods adds to our arsenal of strategies for carbon dioxide capture and use. Support or Funding Information DOE:DE‐FG02‐04ER15563 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Mattice, Jenna↗
DOE OSTI2020

NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?

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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

The Role of Mass Spectrometry in Structural Studies of Flavin-Based Electron Bifurcating Enzymes

For decades, biologists and biochemists have taken advantage of atomic resolution structural models of proteins from X-ray crystallography, nuclear magnetic resonance spectroscopy, and more recently cryo-electron microscopy. However, not all proteins relent to structural analyses using these approaches, and as the depth of knowledge increases, additional data elucidating a mechanistic understanding of protein function is desired. Flavin-based electron bifurcating enzymes, which are responsible for producing high energy compounds through the simultaneous endergonic and exergonic reduction of two intercellular electron carriers (i.e., NAD + and ferredoxin) are one class of proteins that have challenged structural biologists and in which there is great interest to understand the mechanism behind electron gating. A limited number of X-ray crystallography projects have been successful; however, it is clear that to understand how these enzymes function, techniques that can reveal detailed in solution information about protein structure, dynamics, and interactions involved in the bifurcating reaction are needed. In this review, we cover a general set of mass spectrometry-based techniques that, combined with protein modeling, are capable of providing information on both protein structure and dynamics. Techniques discussed include surface labeling, covalent cross-linking, native mass spectrometry, and hydrogen/deuterium exchange. We cover how biophysical data can be used to validate computationally generated protein models and develop mechanistic explanations for regulation and performance of enzymes and protein complexes. Our focus will be on flavin-based electron bifurcating enzymes, but the broad applicability of the techniques will be showcased.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Photoinduced Electron Transfer Informs on Pathway Coupling in Flavin-Based Electron Bifurcation

Flavin-based electron bifurcation (FBEB) is an enzymatic mechanism that generates extremely high-energy electrons to drive unfavorable chemical reactions. It is utilized by the NADHdependent ferredoxin:NADP + -oxidoreductase (Nfn) enzyme in hyperthermophile Pyrococcus furiosus to bifurcate electrons from NADPH into the coupled low-potential (endergonic) and highpotential (exergonic) pathways. This process enables P. furiosus to live in harsh and uninviting environments. Despite its biological importance, the mechanisms used by Nfn to facilitate exceptional directional control over short-lived, high-energy electrons and to prevent undesired transfer, particularly along the low-potential pathway, are still not well understood. To elucidate how the protein environment contributes to electronic control in the lowpotential pathway, new techniques must be utilized to probe these unstable intermediates. In this study, we have adapted lowtemperature photoexcitation combined with electron paramagnetic resonance (EPR) to accumulate the short-lived intermediate and place it in the context of the other cofactors involved in the low-potential pathway of Nfn. We observed coincident growth of both the radical intermediate and its nearby [4Fe-4S] cluster over 4.5 h of illumination with NADPH at cryogenic temperatures. The photogenerated paramagnetic species were stable in LN 2 storage indefinitely and recombined when warmed to higher temperatures. The results provide insights into the electron transfer steps and cofactor interactions along the low potential pathway, facilitating a more robust mechanistic understanding of the high-energy events of electron bifurcation. Furthermore, through comparison of cryogenic and room temperature experiments, a potential gating step involving the movement of key residues important for the reversibility of electron flow along this pathway is suggested.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Photoenzymatic Reductions Enabled by Direct Excitation of Flavin-Dependent “Ene”-Reductases

Non-natural photoenzymatic reactions reported to date have depended on the excitation of electron donor–acceptor complexes formed between substrates and cofactors within protein active sites to facilitate electron transfer. While this mechanism has unlocked new reactivity, it limits the types of substrates that can be involved in this area of catalysis. Here we demonstrate that direct excitation of flavin hydroquinone within “ene”-reductase active sites enables new substrates to participate in photoenzymatic reactions. Here, we found that by using photoexcitation these enzymes gain the ability to reduce acrylamides through a single electron transfer mechanism

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Flavin-dependent halogenases catalyze enantioselective olefin halocyclization

Abstract Halocyclization of alkenes is a powerful bond-forming tool in synthetic organic chemistry and a key step in natural product biosynthesis, but catalyzing halocyclization with high enantioselectivity remains a challenging task. Identifying suitable enzymes that catalyze enantioselective halocyclization of simple olefins would therefore have significant synthetic value. Flavin-dependent halogenases (FDHs) catalyze halogenation of arene and enol(ate) substrates. Herein, we reveal that FDHs engineered to catalyze site-selective aromatic halogenation also catalyze non-native bromolactonization of olefins with high enantioselectivity and near-native catalytic proficiency. Highly selective halocyclization is achieved by characterizing and mitigating the release of HOBr from the FDH active site using a combination of reaction optimization and protein engineering. The structural origins of improvements imparted by mutations responsible for the emergence of halocyclase activity are discussed. This expansion of FDH catalytic activity presages the development of a wide range of biocatalytic halogenation reactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

An uncharacteristically low-potential flavin governs the energy landscape of electron bifurcation

Significance Nature has long been an inspiration for materials design, as it exemplifies exquisite control of both matter and energy. Electron bifurcation, a mechanism employed in biological systems to drive thermodynamically unfavorable and energetically challenging chemical reactions, is one such example. A key feature of bifurcating enzymes is the ability of a single redox cofactor to distribute a pair of electrons across two spatially separated electron transfer pathways. Here, we report on the empirical determination of both the one-electron potential and two-electron potential of the bifurcating flavin cofactor in the NADH-dependent ferredoxin-NADP + oxidoreductase I (NfnSL) enzyme. Insights arising from the defined energy landscape of this bifurcation site may underlie the design of synthetic catalysts capable of generating high-energy intermediates.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction

Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes’ innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity. Here, we investigate the structural features that control the stereoselectivity in an oxidative dearomatization reaction that is key to making azaphilone natural products. Crystal structures of enantiocomplementary biocatalysts guided the development of multiple hypotheses centered on the structural features that control the stereochemical outcome of the reaction; however, in many cases, direct substitutions of active site residues in natural proteins led to inactive enzymes. Ancestral sequence reconstruction (ASR) and resurrection were employed as an alternative strategy to probe the impact of each residue on the stereochemical outcome of the dearomatization reaction. These studies suggest that two mechanisms are active in controlling the stereochemical outcome of the oxidative dearomatization reaction: one involving multiple active site residues in AzaH and the other dominated by a single Phe to Tyr switch in TropB and AfoD. Moreover, this study suggests that the flavin-dependent monooxygenases (FDMOs) adopt simple and flexible strategies to control stereoselectivity, which has led to stereocomplementary azaphilone natural products produced by fungi. This paradigm of combining ASR and resurrection with mutational and computational studies showcases sets of tools for understanding enzyme mechanisms and provides a solid foundation for future protein engineering efforts.

Science & Technology - Other Topics↗