NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV0) of the opening transition. While the ΔV0 characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.
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NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?
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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- Award ID(s):
- 1904480
- NSF-PAR ID:
- 10237202
- Editor(s):
- Pettinari, M. Julia
- Date Published:
- Journal Name:
- Applied and Environmental Microbiology
- Volume:
- 86
- Issue:
- 22
- ISSN:
- 0099-2240
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Lactate monooxygenase (LMO) catalyzes the FMN‐dependent “coupled” oxidation of lactate and O2to acetate, carbon dioxide, and water, involving pyruvate and hydrogen peroxide as enzyme‐bound intermediates. Other α‐hydroxy acid oxidase family members follow an “uncoupled pathway,” wherein the α‐keto acid product quickly dissociates before the reduced flavin reacts with oxygen. Here, we report the structures of
Mycobacterium smegmatis wild‐type LMO and a wild‐type‐like C203A variant at 2.1 Å and 1.7 Å resolution, respectively. The overall LMO fold and active site organization, including a bound sulfate mimicking substrate, resemble those of other α‐hydroxy acid oxidases. Based on structural similarity, LMO is similarly distant from lactate oxidase, glycolate oxidase, mandelate dehydrogenase, and flavocytochrome b2and is the first representative enzyme of its type. Comparisons with other α‐hydroxy acid oxidases reveal that LMO has a longer and more compact folded active site loop (Loop 4), which is known in related flavoenzymes to undergo order/disorder transitions to allow substrate/product binding and release. We propose that LMO's Loop 4 has an enhanced stability that is responsible for the slow product release requisite for the coupled pathway. We also note electrostatic features of the LMO active site that promote substrate binding. Whereas the physiological role of LMO remains unknown, we document what can currently be assessed of LMO's distribution in nature, including its unexpected occurrence, presumably through horizontal gene transfer, in halophilic archaea and in a limited group of fungi of the genusBeauveria .Broad statement of impact This first crystal structure of the FMN‐dependent α‐hydroxy acid oxidase family member lactate monooxygenase (LMO) reveals it has a uniquely large active site lid that we hypothesize is stable enough to explain the slow dissociation of pyruvate that leads to its “coupled” oxidation of lactate and O2to produce acetate, carbon dioxide, and water. Also, the relatively widespread distribution of putative LMOs supports their importance and provides new motivation for their further study.
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Dihydropyrimidine dehydrogenase (DPD) catalyzes the initial step in the catabolism of the pyrimidines uracil and thymine. Crystal structures have revealed an elaborate subunit architecture consisting of two flavin cofactors, apparently linked by four Fe4S4 centers. Analysis of the DPD reaction(s) equilibrium position under anaerobic conditions revealed a reaction that favors dihydropyrimidine formation. Single-turnover analysis shows biphasic kinetics. The serine variant of the candidate general acid, cysteine 671, provided enhanced kinetic resolution for these phases. In the first event, one subunit of the DPD dimer takes up two electrons from NADPH in a reductive activation step. Spectrophotometric deconvolution suggests that thes electrons reside on one of the two flavins. That oxidation of the enzyme by dioxygen can be suppressed by the addition of pyrimidine, is consistent with these electrons residing on the FMN. The second phase involves further oxidation of NADPH and concomitant reduction of the pyrimidine substrate. During this phase no net reduction of DPD cofactors is observed indicating that the entire cofactor set acts as a wire, transmitting electrons from NADPH to the pyrimidine rapidly. This indicates that the availability of the proton from C671 general acid controls the transmittance of electrons from NADPH to the pyrimidine. Acid quench and HPLC product analysis of single-turnover reactions with limiting NADPH confirmed 2:1, NADPH:pyrimidine stoichiometry for the enzyme accounting for successive activation and pyrimidine reduction. These data support an alternating subunit model in which one protomer is activated and turns over before the other subunit can be activated and enter catalysis.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/AEM.01758-20
