Thioredoxin reductase from Bacillus cereus exhibits distinct reduction and NADPH-binding properties.

Low-molecular-weight (low Mr ) thioredoxin reductases (TrxRs) are homodimeric NADPH-dependent dithiol flavoenzymes that reduce thioredoxins (Trxs) or Trx-like proteins involved in the activation networks of enzymes, such as the bacterial class Ib ribonucleotide reductase (RNR). During the last few decades, TrxR-like ferredoxin/flavodoxin NADP+ oxidoreductases (FNRs) have been discovered and characterized in several types of bacteria, including those not encoding the canonical plant-type FNR. In Bacillus cereus, a TrxR-like FNR has been shown to reduce the flavodoxin-like protein NrdI in the activation of class Ib RNR. However, some species only encode TrxR and lack the homologous TrxR-like FNR. Due to the structural similarity between TrxRs and TrxR-like FNRs, as well as variations in their occurrence in different microorganisms, we hypothesized that low Mr TrxR may be able to replace TrxR-like FNR in, for example, the reduction of NrdI. In this study, characterization of TrxR from B. cereus has revealed a weak FNR activity toward NrdI reduction. Additionally, the crystal structure shows that only one out of two binding sites of the B. cereus TrxR homodimer is occupied with NADPH, indicating a possible asymmetric co-substrate binding in TrxR.

The Crystal Structures of Bacillithiol Disulfide Reductase Bdr (YpdA) Provide Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductase.

Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial for maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely, those from S. aureus and B. cereus. Our analyses reveal a uniquely organized biological tetramer; however, the structure of the monomeric subunit is highly similar to those of other flavoprotein disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in the proximity of the FAD cofactor. We also report enzymatic activities for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.

The Characterization of Different Flavodoxin Reductase-Flavodoxin (FNR-Fld) Interactions Reveals an Efficient FNR-Fld Redox Pair and Identifies a Novel FNR Subclass.

Flavodoxins (Flds) are small, bacterial proteins that transfer electrons to various redox enzymes. Flavodoxins are reduced by ferredoxin/flavodoxin NADP+ oxidoreductases (FNRs), but little is known of the FNR-Fld interaction. Here, we compare the interactions of two flavodoxins (Fld1-2), one flavodoxin-like protein (NrdI), and three different thioredoxin reductase (TrxR)-like FNRs (FNR1-3), all from Bacillus cereus. Steady-state kinetics shows that the FNR2-Fld2 electron transfer pair is particularly efficient, and redox potential measurements also indicate that this is the most favorable electron donor/acceptor pair. Furthermore, crystal structures of FNR1 and FNR2 show that the proteins have crystallized in different conformations, a closed and an open conformation, respectively. We suggest that a large-scale conformational rearrangement takes place during the FNR catalytic cycle to allow for the binding and reduction of the Fld and, subsequently, the re-reduction of the FNR by NADPH. Finally, inspection of the residues surrounding the FAD cofactor in the FNR active site shows that a key isoalloxazine ring-stacking residue is different in FNR1 and FNR2, which could explain the large difference in catalytic efficiency between the two FNRs. To date, all of the characterized TrxR-like FNRs have a residue with aromatic character stacking against the FAD isoalloxazine ring, and this has been thought to be a conserved feature of this class of FNRs. FNR1, however, has a valine in this position. Bioinformatic analysis shows that the TrxR-like FNRs can actually be divided into two groups, one group where the FAD-stacking residue has aromatic character and another group where it is valine.

High-resolution crystal structures reveal a mixture of conformers of the Gly61-Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus.

Flavodoxins (Flds) are small proteins that shuttle electrons in a range of reactions in microorganisms. Flds contain a redox-active cofactor, a flavin mononucleotide (FMN), and it is well established that when Flds are reduced by one electron, a peptide bond close to the FMN isoalloxazine ring flips to form a new hydrogen bond with the FMN N5H, stabilizing the one-electron reduced state. Here, we present high-resolution crystal structures of Flavodoxin 1 from Bacillus cereus in both the oxidized (ox) and one-electron reduced (semiquinone, sq) state. We observe a mixture of conformers in the oxidized state; a 50:50 distribution between the established oxidized conformation where the peptide bond is pointing away from the flavin, and a conformation where the peptide bond is pointing toward the flavin, approximating the conformation in the semiquinone state. We use single-crystal spectroscopy to demonstrate that the mixture of conformers is not caused by radiation damage to the crystal. This is the first time that such a mixture of conformers is reported in a wild-type Fld. We therefore carried out a survey of published Fld structures, which show that several proteins have a pronounced conformational flexibility of this peptide bond. The degree of flexibility seems to be modulated by the presence, or absence, of stabilizing interactions between the peptide bond carbonyl and its surrounding amino acids. We hypothesize that the degree of conformational flexibility will affect the Fld ox/sq redox potential.

Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST).

This protocol describes how to measure protein-protein interactions by microscale thermophoresis (MST) using the MonolithTM NT.115 instrument (NanoTemper). We have used the protocol to determine the binding affinities between three different flavodoxin reductases (FNRs) and a flavodoxin-like protein, NrdI, from Bacillus cereus ( Lofstad et al., 2016 ). NrdI is essential in the activation of the manganese-bound form of the class Ib ribonucleotide reductase (RNR) system. RNRs, in turn, are the only source of the de novo synthesis of deoxyribonucleotides required for DNA replication and repair in all living organisms.

Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.

To reduce ribonucleotides to deoxyribonucleotides, the manganese-bound form of class Ib ribonucleotide reductase (RNR) must be activated via a pathway that involves redox protein(s). The reduced flavoprotein NrdI is an important protein in this pathway, as it reduces dioxygen to superoxide. Superoxide then reacts with the RNR Mn(II)2 site to generate a tyrosyl radical that is required for catalysis. A native NrdI reductase has not yet been identified. We herein demonstrate through kinetic and spectroscopic studies that an endogenous flavodoxin reductase can function as the NrdI reductase in Bacillus cereus. When the flavodoxin reductase reduces NrdI, tyrosyl radical formation in RNR is promoted under aerobic conditions, significantly increasing the radical yield. Thus, a missing piece of the class Ib RNR NrdI redox pathway has finally been identified.

Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O2 and CO.

The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H2-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H2. The H2 oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O2 and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site "gate" gas access, preventing molecules larger than H2 interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O2 is in fact an inhibitor of H2 oxidation by the RH, and that both H(+) reduction and H2 oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O2 arises due to interaction of O2 with the active site. Using protein film infrared electrochemistry (PFIRE) under H2 oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases.