Archives of Biochemistry and Biophysics 702 (2021) 108826

Contents lists available at ScienceDirect

Archives of Biochemistry and Biophysics

journal homepage: www.elsevier.com/locate/yabbi

Overview of structurally homologous flavoprotein containing the low Mr -like fold – A functionally diverse group

Marta Hammerstad a,**, Hans-Petter Hersleth a,b,* a Department of Biosciences, University of Oslo, Section for Biochemistry and Molecular Biology, P.O. Box 1066, Blindern, NO-0316, Oslo, Norway b Department of Chemistry, University of Oslo, Section for Chemical Life Sciences, University of Oslo, P.O. Box 1033, Blindern, NO-0315, Oslo, Norway

ARTICLE INFO ABSTRACT

Keywords: Structural studies show that have a limited number of unique folds, although structurally related en­ zymes have evolved to perform a large variety of functions. In this review, we have focused on enzymes con­ Flavoprotein taining the low molecular weight thioredoxin reductase (low Mr TrxR) fold. This fold consists of two domains, Thioredoxin reductase both containing a three-layer ββα sandwich Rossmann-like fold, serving as flavin adenine dinucleotide (FAD) Structure and, in most cases, pyridine nucleotide (NAD(P)H) binding-domains. Based on a search of the Protein Data Bank Pyridine nucleotide for all published structures containing the low Mr TrxR-like fold, we here present a comprehensive overview of Flavin adenine dinucleotide + Redox enzymes with this structural architecture. These range from TrxR-like ferredoxin/flavodoxin NAD(P) oxido­ reductases, through , to NADH peroxidase. Some enzymes are solely composed of the low Mr TrxR-like fold, while others contain one or two additional domains. In this review, we give a detailed description of selected enzymes containing only the low Mr TrxR-like fold, however, catalyzing a diversity of chemical reactions. Our overview of this structurally similar, yet functionally distinct group of flavoprotein oxidoreductases highlights the fascinating and increasing number of studies describing the diversity among these enzymes, especially during the last decade(s).

1. Flavoproteins and flavoenzymes electron transfer reactions, oxygen activation, and photochemistry. Ex­ amples of enzymatic reactions catalyzed by flavoenzymes include Flavins are versatile cofactors due to the reactivity and multiple monooxygenation, dehydrogenation, and halogenation reactions, taking oxidation states of the redox-active isoalloxazine ring system (Fig. 1A). place in various biological processes such as DNA repair, oxidative Because of the various oxidation states (oxidized (ox), semiquinone (sq), protein folding, and countless biosyntheses [3,6]. Due to their selectivity and hydroquinone (hq)), flavins are able to perform both one-electron and efficiency, flavoenzymes are attractive for use in industrial bio­ and two-electron transfer reactions; a reactivity associated to the , metabolic engineering, and synthetic biology. The largest flavinisoalloxazine ring, focused on the reactive N5 and C4a atoms [1,2] group of flavin-dependent enzymes belong to the oxidoreductases (Fig. 1A). For most flavoproteins, the flavin cofactor is either a flavin ( Commission (EC) class 1) (91%) [3]. The focus of this review mononucleotide (FMN) or a flavin adenine dinucleotide (FAD), will be on structural aspects of specific flavin-dependent non-covalently bound to the protein [3] (Fig. 1A). The majority of fla­ oxidoreductases. voenzymes utilize FAD (75%) rather than FMN (25%), predominantly bound in a Rossmann-like fold; the three-layer αβα (CATH superfamily 2. Key oxidoreductases of the “two dinucleotide binding 3.40.50.x) or the ββα (CATH superfamily 3.50.50.60) sandwich fold domains” flavoprotein (tDBDF) superfamily [3–5]. Flavoenzymes are involved in a wide and diverse range of bio­ logical processes, carrying out reductions and oxidations in Oxidoreductases, comprising almost one third of all enzymatic

* Corresponding author. Department of Biosciences, University of Oslo, Section for Biochemistry and Molecular Biology, P.O. Box 1066, Blindern, NO-0316, Oslo, Norway. ** Corresponding author. E-mail addresses: [email protected] (M. Hammerstad), [email protected] (H.-P. Hersleth). https://doi.org/10.1016/j.abb.2021.108826 Received 13 November 2020; Received in revised form 23 February 2021; Accepted 27 February 2021 Available online 5 March 2021 0003-9861/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Fig. 1. Structures and redox states of cofactors in flavoprotein oxidoreductases. (A) FMN and FAD, showing the isoalloxazine ring in oxidized (ox), semi­ + + quinone (sq), and hydroquinone (hq) states, (B) NADPH/NADP and NADH/NAD , (C) Redox-active disulfide/thiols. The reactive C4a and N5 atoms of the isoalloxazine ring are highlighted in red. (For interpretation of the references to color in this figurelegend, the reader is referred to the Web version of this article.) activities registered in the BRaunschweig ENzyme Database (BRENDA) thiols (e.g. glutathione (GSH)) [10,11]. The majority of flavoprotein [7], catalyze the oxidation of one chemical species (reducing agent/e­ disulfide reductases (FDRs) belong to oxidoreductase subgroup EC 1.8. lectron donor) coupled to the reduction of another (oxidizing agent/e­ FDRs represent a family of enzymes with high sequence and structural lectron acceptor). Generally, the reduction/oxidation of a substrate is homology, catalyzing the NAD(P)H-dependent reduction of coupled to the oxidation/reduction of a nicotinamide adenine dinucle­ sulfide-bonded substrates. This group of enzymes include e.g. thio­ + otide cofactor NAD(P)H/NAD(P) (Fig. 1B). NAD(P)H-dependent oxi­ redoxin reductase (TrxR) (EC 1.8.1.9), glutathione reductase (GR) (EC doreductases catalyze a wide range of central biological reactions, 1.8.1.7), dihydrolipoamide dehydrogenase (DLD) (EC 1.8.1.4), trypa­ acting on both organic and inorganic substrates [8]. Many NAD(P) nothione reductase (TryR) (EC 1.8.1.12), and mycothione reductase H-dependent oxidoreductases, like alcohol dehydrogenases, transfer (Mtr) (EC 1.8.1.15). Representatives of FDRs are also classifiedin other electrons directly between the substrate and the nicotinamide cofactor, EC subgroups, such as mercuric reductase (MerA) (EC 1.16.1.1) and while many other require additional intermediate cofactors such as NADH peroxidase (NPX) (EC 1.11.1.1) [8,12–14]. flavins, metallocofactors, or redox-active cysteine pairs (Fig. 1C). In The largest subgroup of flavin-dependent oxidoreductases consti­ FAD-containing NAD(P)H-dependent oxidoreductases, namely the “two tutes enzymes performing oxygenation and monooxygenation reactions dinucleotide binding domains” flavoproteins(tDBDF), two dinucleotide (oxygenases/monooxygenases) (EC 1.13 and EC 1.14) [3,8], which also binding Rossmann-like fold domains are fused in a single chain. Each of comprise a number of tDBDFs. Although characterization of mono­ these domains bind one of the two dinucleotide cofactors, FAD and a oxygenases has revealed a number of different enzymes utilizing other pyridine nucleotide, predominantly in the N-terminal and C-terminal cofactors, e.g. heme-containing cytochrome P450 monooxygenases (EC domain, respectively [9]. 1.14.13, EC 1.14.14, and EC 1.14.15) and copper-dependent mono­ A large amount of tDBDFs are found within the diverse class oxygenases (EC 1.14.17 and EC 1.14.18), a large and ubiquitous group comprising oxidoreductases acting on sulfur (EC 1.8). The sulfur- of monooxygenases is formed by the flavin-dependent enzymes [15]. containing group of the substrate can vary greatly within this class, The specifictype of oxygenation reactions and selectivity depends on the and may include inorganic forms and a variety of sulfur-containing nature of the of each specificmonooxygenase, however, also organic molecules such as cysteines and low-molecular weight (LMW) on their structural fold. External flavoprotein monooxygenases

2 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Fig. 2. Structural features of TrxRs. (A) Crystal structure of the low Mr TrxR homodimer in the FO form (PDBid:1TDE, NADPH coordinates from 1TDF), showing the NADPH-binding domain and FAD-binding domain in purple and green, respec­ tively. The two domains are connected by an antiparallel β-sheet hinge region. Both domains contain a three-layer ββα sandwich Rossmann-like fold. Conserved binding motifs for FAD and NADPH are indicated with arrows. (B) The low Mr TrxR active site, with the NADPH and FAD co­ factors, as well as the redox-active cysteine pair, represented as sticks and colored by atom type. The cystine is located close to the FAD cofactor, on the re-face of the isoalloxazine ring (FO form). (C) Crystal structure of the high Mr TrxR homodimer (PDBid:3EAO, NADPH coordinates from 2ZZC), showing the overall fold with the NADPH-binding domain, FAD-binding domain, and interface domain (blue). Cofactors and redox-active motifs are represented as sticks and colored by atom type, and indicated with arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

(consuming NADPH or NADH for flavin reduction) have been grouped iron-sulfur (Fe–S) proteins and of flavodoxin (Fld) [8]. Ferredoxin/­ + according to sequence and structural data [15]. Subclass B flavoprotein flavodoxin NAD(P) oxidoreductases (FNRs) catalyze the electron monooxygenases (FPMOs) comprise three sequence-related subfamilies; transfer between NAD(P)H and the Fe–S cluster of ferredoxin (Fd) or Fld, microbial N-hydroxylating monooxygenases (NMOs) (EC 1.14.13.59), a small, FMN-containing protein which participates in several redox Baeyer-Villiger monooxygenases (Type I BVMOs) (EC 1.14.13.22), and reactions, and which can replace Fd under low iron availability. In flavin-containing monooxygenases (FMOs) (EC 1.14.13.8) [1,6,15]. photosynthetic organisms and tissues, FNR receives electrons from + FMO from Stenotrophomonas maltophilia, SmFMO, catalyzes the asym­ Photosystem I via Fd, and reduces NADP to NADPH [18,19]. metric oxidation of thioethers, and is structurally very similar to e.g. Conversely, FNRs can reduce Fds or Flds with electrons from NADPH. TrxR [16,17]. Fds and Flds can in turn donate electrons to various redox enzymes, such Members of the tDBDF superfamily are also found within oxidore­ as cytochrome P450 (Fd) [20] or nitric oxide synthase (NOS) (Fld) [21]. ductase subgroups EC 1.18 and 1.19, representing oxidoreductases of FNRs can be divided into two main structurally unrelated families,

Fig. 3. Low Mr TrxR structure and mechanism. (A) Structural overview of the domain rotation in TrxR during catalysis, going from the FO conformation ◦ (reduction of the disulfideby FAD) to the FR conformation (reduction of FAD by NADPH and reduction of Trx). The conformational change involves a ~67 rotation of the NADPH domain. FO (PDBid:1TDE and 1TDF, NADPH from the latter), FR (PDBid:1F6M), and the two middle structures are interpolated trajectories (morph) between the FO and FR structures generated by PyMOL. (B) Reaction mechanism of Trx reduction by TrxR.

3 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826 plant-type FNRs and GR-like FNRs. The latter can be further divided into this conformation, however, NADPH binds far from the flavincofactor, adrenodoxin reductase (AdR)-like FNRs, oxygen-coupled NAD(P) and the active site cysteines are buried and cannot react with the Trx ◦ H-ferredoxin reductase (ONFR)-like FNRs, and TrxR-like FNRs. As the substrate. Therefore, a conformational change occurs, with a ~67 name implies, FNRs from the latter subgroup are sequence-wise and rotation of the NADPH domain in respect to the FAD domain (FR, flavin structurally very similar to TrxRs, however, lacking the TrxR catalytic reducing), permitting reduction of FAD by NADPH, and moving the redox-active CXXC-motif/Cys-pair responsible for the reduction of the dithiol pair to the hydrophilic surface of the enzyme to enable thioredoxin (Trx) substrate [13,22–24]. dithiol-disulfide interchange with Trx [33,35,36]. In more detail, the mechanism used by low Mr TrxR can be described as two half-reactions 3. Thioredoxin reductase (TrxR) – NADPH-dependent (Fig. 3B). In the reductive half-reaction, the oxidized flavinis reduced by flavoprotein disulfide oxidoreductase the NADPH hydride, and the reduced flavin enediamine attacks the enzyme disulfide forming a flavin C4a-cysteine adduct and expelling a The structure and fold of TrxR has set the basis of this work. In this thiol. Formation of the thiol is assisted by an active site acid (Asp in section, TrxR classification, structure, redox partner, and mechanism E. coli TrxR) [37]. Then, elimination of the thiolate from the C4a-adduct will be described. TrxR reduces Trx. The latter is a small (12 kDa) results in flavinreoxidation and generation of the reduced thiol-thiolate dithiol-containing protein involved in a variety of cellular processes pair that represents the two-electron reduced state of the enzyme. The such as DNA synthesis and repair, redox signaling, apoptosis, and anti­ thiolate near C4a forms a charge-transfer complex with the oxidized oxidant defense [25–28]. TrxR is a homodimeric flavoprotein (EC flavin. In the oxidative half-reaction, a series of thiol–disulfide in­ 1.8.1.9) catalyzing the NADPH-dependent reduction of the disulfide of terchanges take place between the enzyme redox-active thiols and the Trx to a dithiol [13]. Low molecular weight (low Mr) TrxRs (~35 kDa for oxidizing Trx substrate. First, the interchange thiol (not involved in each subunit) (Fig. 2A and B), which are found in bacteria (Escherichia charge-transfer with the flavin) attacks the Trx disulfide, expelling a coli and others), archaea (Methanococcus jannaschii), plants (Arabidopsis thiolate leaving group, which is protonated by an active site acid. The thaliana) and some lower eukaryotes (yeast, mycoplasmas, Guardia enzyme–substrate mixed disulfide is then attacked by the duodenalis) are distinguished from high molecular weight (high Mr) charge-transfer thiolate, reforming the enzyme disulfide and yielding TrxRs (~55 kDa for each subunit) (Fig. 2C), generally present in higher reduced Trx [38]. eukaryotes (mammals, Plasmodium falciparum, Caenorhabditis elegans, The crystal structure of the FO form of E. coli TrxR was solved by and Drosophila melanogaster) [13,29]. Both types of TrxRs use the same Model and coworkers in 1991 (PDBid:1TRB) [39], followed by addi­ basic chemistry for Trx reduction, however, the details of the reactions tional structures solved by Kuriyan and coworkers in 1994 are different, where high Mr TrxRs, in addition to having an enzymatic (PDBid:1TDE,1TDF) [36], who also presented models of the alternative disulfidenear the FAD group on the si-face (re-face in low Mr TrxR), have FR form. Ludwig and coworkers determined the structure of the E. coli a second redox-active disulfide/selenosulfide pair at their extended TrxR FR form in 2000 (PDBid:1F6M) [33], providing evidence for the C-terminus [12,13,29,30] (Fig. 2 and Section 4.3). In high Mr TrxRs, the conformational changes required for catalysis. The latter structure was electrons are donated from NADPH on the re-face of FAD to the disul­ obtained from a crystal complex of cross-linked TrxR with Trx. fides located on the FAD si-face and further to the disul­ The structure of low Mr TrxR is conserved, with varying degrees of fide/selenosulfide pair on the other monomer, which reduces the modifications, among numerous oxidoreductases. Throughout evolu­ disulfide pair in Trx (Fig. 2C and Section 4.3). Structurally, each tion, these structurally related enzymes have beautifully diverged to monomer of the high Mr TrxR contains a FAD-binding domain, a perform multiple cellular processes. This structural basis has set the NADPH-binding domain, and an interface or dimerization domain background for this review, including a comprehensive structural (Fig. 2C). High Mr TrxRs will not be further discussed in this review. analysis of proteins containing the low Mr TrxR-like fold. Low Mr TrxR contains FAD and NADPH-binding domains, with the redox-active disulfidelocated in the conserved active site CXXC motif in 4. Structural analysis of proteins containing the low Mr TrxR- the pyridine-nucleotide-binding domain. The two globular domains, like fold – a tDBDFs subclass both containing a three-layer ββα sandwich Rossmann-like fold, are connected by a two-stranded β-sheet (Fig. 2A). Conserved amino acid Oxidoreductases represent a numerous and functionally diverse class sequence motifs responsible for the binding of FAD and NAD(P)H are of enzymes. Oxidoreductases from many subclasses, however, share located in the two binding domains, respectively, which is a common high structural similarity, and in particular the NAD(P)H-dependent feature among TrxRs and many TrxR-like oxidoreductases utilizing FAD oxidoreductases (50% of all oxidoreductases) [8]. The overall struc­ ′ and NAD(P)H as cofactors (HRRXXXR, 2 P phosphate group of NADPH; ture composed of two dinucleotide-binding domains, responsible for GXGXXA/G, pyrophosphate group of NAD(P)H); GXGXXG, FAD) binding FAD and NAD(P)H, respectively, is seen in many of these oxi­ (Fig. 2). The identity of these sequence motifs may vary among different doreductases, and is commonly described for the low Mr TrxRs, typified classes of FAD- and NAD(P)H-dependent oxidoreductases [31,32]. In by the E. coli enzyme [35]. Some flavin-dependent oxidoreductases, ′ particular, the motif responsible for binding of the 2 P phosphate group together with low Mr TrxR, are composed solely of the low Mr TrxR of NADPH can be absent or altered in oxidoreductases shown to bind and architecture, but with different orientations of the dinucleotide-binding utilize NADPH. Amino acid substitutions of residues in the typical domains relative to each other. Also, an increasing number of oxidore­ HRRXXXR motif occur in several NADPH-consuming oxidoreductases, ductases have been described which are composed solely of the low Mr although they are typically replaced with other motifs containing basic TrxR architecture, however, functioning in an NAD(P)H-independent ′ amino acids interacting with the 2 P phosphate group of the adenosine. manner. Other oxidoreductases may contain the low Mr TrxR architec­ The catalysis of low Mr TrxR, unlike what is seen for high Mr TrxRs ture, however, with additional domains present, making up larger [30], involves a large conformational change where the NADPH domain structures, as will be discussed below. To get an overview of all struc­ ◦ rotates ~67 relative to the FAD domain. This domain rotation is tures available containing the low Mr TrxR-like fold, we searched the necessary in order to sequentially accommodate NADPH and the Protein Data Bank (PDB) by performing a DALI (Distance-matrix redox-active disulfide close to the isoallozaxine ring of FAD (on the ALIgnment) search [40], using the E. coli low Mr TrxR structures as re-face), and to expose the reduced dithiol to the Trx substrate [33,34] search templates. From our structural alignment search, we inspected (Fig. 3A). In one conformation (FO, flavin oxidizing), the loop con­ the thousand most similar hits containing the low Mr TrxR fold, to taining the redox-active disulfideadjoins the re-face of the FAD cofactor identify unique protein types. Some hits were excluded since they con­ in an orientation allowing reduction of the disulfide by the reduced tained only the FAD-binding domain (e.g. pyranose 2-oxidase and flavin, forming an oxidized flavin-thiolate charge transfer complex. In lysine-specific histone demethylase), or had a FAD-binding domain

4 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826 example 3EF6 4H4W 5FS9 6KRT 2V3A 5ODQ 3T2Z 6OEX 1ZK7 1Q1W 1F8W 5ER0 4EQX 6PFZ 3EAO 1GRB 2R9Z 2EQ9 5W1J 1TDF 6GAS 5TWC 7A7B 4NTC,4NTD, 4NTE 5J60,5NII 6GNB 5N0J 7BR0 1HYU 4USR 3S5W 3GWD,2YLS, 4AOX PDB steroid holomycin, 2 I thiols 10 Se, 2 phenylacetone, gliotoxin, CoQ H Pdx P, small , of disulfide Fd Fd S, 2 Rdx of A S peroxide , N, S-CoM, factors – S HS (Fld) – + Trx 2 S, 2 putidaredoxin Rieske-type Rieske-type import monodehydroascorbate rubredoxin hydrogen dioxygen coenzyme persulfides GSSG/Trx Trx GSSG trypanothione/Try[S] GASSAG dihydrolipoamide Hg H Trx Fld/Fd iron-siderophore/Isdl/IsdG BSSB Biosynthesis romidepsin Trx, Fd, thiols/unknown prespiro-aspirochlorine AhpC C-bound ornithine cyclohexanone, CoB Substrate/Partner + + + + + + + + + + + + + + + + + - + + + + - - - - - + + + + - NAD(P) H C, 2 Cys CSec CX CC, Cys Cys Cys -C C, C C C, C, C n 4 4 4 4 4 2 single CXXC C-X CX No No No No No No single single single 2 CXXC CXXC CX CX CX Grx CX CXXC CX No No No CXXC CXXC CXXC CXXC CXXH 2 No No No No Active cysteines domain of anchor N-linker rhodanese Grx NmerA domain domain part - motif fold membrane domain domain domain domain, domain domains Rdx-binding domain domain domain domain domain, domain domain domain domain, domain, domain Trx N-terminal C-terminal interface C-terminal C-terminal C-terminal C-terminal C-terminal C-terminal interface interface interface interface domain interface interface interface interface interface - interface ------double helix-loop-helix substrate-binding substrate-binding 2Fd, complex Additional fold. TrxR-like TRi HlmI, (low) (high) r M SQR GAR Pdr TodA BphA4 AIF MDHAR RdxR NPX NOX CoADR Npsr GR TryR DLD TGR MerA TrxR TrxR FNR IruO Bdr GliT, DepH DTR, FFTR DDOR AclR AhpF FMO NMO/PvdA BVMO HdrA Abbreviation low the steroid (3) (5) and containing Cys pair(s) (4) Cys single pair database (2) and and phenylacetone Cys PDB no hydroxylase) the domain domain but reductase AclR P450 in F (1) of subunit A fold types (ornithine interface domain, interface segments/domains dioxygenase protein (cyclohexanone, r subunit oxidoreductase r core) M oxidoreductases + thioredoxin M fold subunit component reductase toluene - reductase oxidoreductase protein high low TrxR-like reductase NADP - - r flavin of Fd reductase additional C-terminal C-terminal C-terminal reductase reductase M factor (catalytic reductase dehydrogenase monooxygenase specialized oxidoreductases monooxygenase low oxidoreductase reductase, disulfide monooxygenases reductase reductase different oxidoreductase disulfide reductase glutathione oxidoreductase reductase the amide reductase reductase disulfide A component additional additional additional reductase the reductase dioxygenase substrate an an an miscellaneous only of peroxidase oxidase hydroperoxide utilizing 1A Sulfide:quinone Thioredoxin Glutathione Putidaredoxin Reductase Biphenyl Apoptosis-inducing Monodehydroascorbate Rubredoxin NADH NADH Coenzyme Persulfide Glutathione Trypanothione Dihydrolipoamide Thioredoxin Mercuric Ferredoxin/flavodoxin Iron Bacillithiol Natural Thioredoxin-like Ferredoxin-dependent Diflavin-linked Thioredoxin Alkyl Flavin-containing N-hydroxylating Baeyer-Villiger Heterodisulfide Thioredoxin monooxygenase) Contains Contains Contains Contains Protein Contains Table Overview

5 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Table 1B Overview of the different protein types in the PDB database containing the low Mr TrxR-like fold – Scores from the DALI search relative to low Mr TrxR (1TDF). Protein Abbreviation Z- RMSD Residues Aligned Sequence PDB score (Å) residues identity (%)

Contains only the low Mr TrxR-like fold (1) Thioredoxin reductase - low Mr TrxR (low) 52.6 0.0 315 315 100 1TDF + Ferredoxin/flavodoxin NADP oxidoreductase FNR 24.5 4.7 326 206 22 6GAS Iron utilizing oxidoreductase IruO 26.6 5.5 344 289 22 5TWC Bacillithiol disulfide reductase Bdr 20.5 3.8 323 235 16 7A7B Natural substrate specialized oxidoreductases GliT, HlmI, 29.5 2.7 308 288 20 4NTC,4NTD, DepH 4NTE Thioredoxin-like oxidoreductases DTR, TRi 27.1 4.7 307 194 31 5J60,5NII Ferredoxin-dependent flavin thioredoxin reductase FFTR 24.9 5.4 284 195 24 6GNB Diflavin-linked disulfide oxidoreductase DDOR 31.3 2.6 300 290 20 5N0J Thioredoxin oxidoreductase fold protein AclR AclR 27.9 2.9 318 288 17 7BR0 Contains miscellaneous additional segments/domains (2) Alkyl hydroperoxide reductase subunit F AhpF 36.8 2.2 521 304 35 1HYU Flavin-containing monooxygenase FMO 17.2 3.7 357 216 15 4USR N-hydroxylating monooxygenase (ornithine hydroxylase) NMO/PvdA 16.4 4.2 411 189 17 3S5W Baeyer-Villiger monooxygenases (cyclohexanone, BVMO 12.4 3.9 529 218 10 3GWD,2YLS, phenylacetone and steroid monooxygenase) 4AOX Heterodisulfide reductase, subunit A HdrA 21.8 4.0 652 228 14 5ODQ Sulfide:quinone oxidoreductase SQR 16.0 3.4 427 212 14 3T2Z Contains an additional C-terminal interface domain and Cys pair(s) (3) Thioredoxin reductase - high Mr TrxR (high) 19.3 4.6 489 235 17 3EAO Glutathione reductase GR 20.6 4.0 461 233 18 1GRB Trypanothione reductase TryR 17.7 3.8 485 231 13 6OEX Glutathione amide reductase GAR 20.2 4.7 451 229 21 2R9Z Dihydrolipoamide dehydrogenase DLD 20.0 6.0 460 252 23 2EQ9 Thioredoxin glutathione reductase TGR 17.6 5.0 584 234 18 5W1J Mercuric reductase (catalytic core) MerA 19.2 3.8 467 220 21 1ZK7 Contains an additional C-terminal domain, but no Cys pair (4) Putidaredoxin reductase - component of P450 Pdr 17.2 3.8 421 218 16 1Q1W Reductase component of toluene dioxygenase TodA 17.8 3.8 400 215 18 3EF6 Biphenyl dioxygenase Fd reductase subunit BphA4 17.3 3.8 399 214 18 4H4W Apoptosis-inducing factor AIF 18.3 3.7 459 218 15 5FS9 Monodehydroascorbate reductase MDHAR 19.7 4.3 433 226 26 6KRT Rubredoxin reductase RdxR 16.3 4.1 381 215 18 2V3A Contains an additional C-terminal interface domain and single Cys (5) NADH peroxidase NPX 18.1 3.5 447 212 22 1F8W NADH oxidase NOX 19.7 4.4 450 211 18 5ER0 Coenzyme A disulfide reductase CoADR 16.8 4.0 437 223 17 4EQX Persulfide reductase Npsr 18.0 3.5 536 220 17 6PFZ

Average used for lines with more than one PDBid. composed of a three-layer αβα sandwich Rossmann-like fold (e.g. dihy­ the low Mr TrxR-like fold is the difference in orientation of the NAD(P)H- droorotate dehydrogenase and AdR) instead of the three-layer ββα binding domain (or pseudo-NAD(P)H-binding domain for NAD(P)H- sandwich Rossmann-like fold found in low Mr TrxR. Based on these independent oxidoreductases) relative to the FAD-binding domain criteria, we identified ~30 unique oxidoreductases with homologous (Table 1A and Fig. 4A–J). These enzymes are naturally the ones with the structural organization, however, with different degrees of structural highest Z-score from the structural similarity search by DALI (Table 1B). similarity (Table 1 and Fig. 4). The identified oxidoreductases have a This group can be divided into the NAD(P)H-dependent (Fig. 4A–E) and great variety of substrates upon which they can react, and variations in NAD(P)H-independent (Fig. 4F–J) oxidoreductases. The active sites of their structural organization. However, the low Mr TrxR-like fold serves the NAD(P)H-dependent enzymes (Fig. 5B,C,F) resemble the active site as a conserved consensus structural feature seen for these enzymes, of other oxidoreductase groups (Fig. 5). However, the activity only oc­ regardless of their catalytic function (Fig. 4). Some oxidoreductases are curs at the re-face of the isoalloxazine ring. For low Mr TrxR, a domain solely composed of the low Mr TrxR-like fold; however, others contain rotation (discussed in Section 3) allows both the redox-active disulfide various numbers of additional domains. Based on our structural survey, and the NAD(P)H to sequentially come close to the isoalloxazine ring of we have grouped these oxidoreductases according to structural simi­ FAD on the re-face (Fig. 5B and C). Similarly, FNR provides open access larity and degree of variation into five groups: (1) oxidoreductases to its redox partner on the FAD re-face [41]. In contrast, except for low containing only the low Mr TrxR-like fold, (2) oxidoreductases con­ Mr TrxR, FNR and N-hydroxylating monooxygenase (NMO), all the other taining the low Mr TrxR-like fold and miscellaneous additional seg­ enzyme active sites presented in Fig. 5 show that the flow of electrons ments/domains, (3) oxidoreductases containing the low Mr TrxR-like goes from NAD(P)H on the re-face of the isoalloxazine ring, to the fold, an additional C-terminal interface domain, and Cys pair(s), (4) cysteine(s) and/or substrates on the si-face of FAD (Fig. 5A,D,G,H,I). The oxidoreductases containing the low Mr TrxR-like fold and an additional main focus of this review will be on the diversity of the members of C-terminal domain, but no Cys pair, and (5) oxidoreductases containing structural group (1) oxidoreductases, with respect to variations in re­ the low Mr TrxR-like fold, an additional C-terminal interface domain, actions, domain flexibility,and rotation. Selected enzymes of this group and an active single Cys. These five structural groups will be described will be further discussed in detail in Sections 5 and 6. briefly below.

4.2. Oxidoreductases containing the low Mr TrxR-like fold and 4.1. Oxidoreductases containing only the low Mr TrxR-like fold (1) miscellaneous additional segments/domains (2)

The most striking structural feature of the enzymes containing only Group 2 is a gathering of low Mr TrxR-like fold containing

6 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Fig. 4. Overview and alignment of the oxidoreductases containing the low Mr TrxR-like fold described in Table 1. The low Mr TrxR-like fold colored in green for the FAD domain and purple for the NAD(P)H domain. Additional C-terminal domain colored in blue, a second C-terminal domain in teal, an N-terminal domain in yellow, an internal domain in orange, and additional helices of the NAD(P)H or FAD domains in light purple or light green, respectively. The cofactors are represented as sticks and colored by atom type. The structures are (A) low Mr TrxR in FO form (PDBid:1TDE and ITDF, NADPH from the latter), (B) low Mr TrxR in FR form (PDBid:5VT3), (C) FNR2 (PDBid:6GAS), (D) IruO (PDBid:5TWC), (E) Bdr (PDBid:7A7B), (F) Overlay of GliT (PDBid:4NTC), HlmI (PDBid:4NTD), and DepH (PDBid:4NTE), (G) Overlay of DTR (PDBid:5J60) and TRi (PDBid:5NII), (H) FFTR (PDBid:6GNB), (I) DDOR (PDBid:5N0J), (J) AclR (PDBid: 7BR0), (K) AhpF (PDBid:1HYU), (L) Overlay of FMO (PDBid:4USR) and NMO/PvdA (PDBid:3S5W), (M) overlay of three BVMOs (PDBid:3GWD, 2YLS and 4AOX), (I) (N) HdrA (PDBid:5ODQ), (O) SQR (PDBid:3T2Z), (P) high Mr TrxR (PDBi­ d:3EAO), (Q) Overlay of GR (PDBid:1GRB), TryR (PDBid:6OEX), GAR (PDBid:2R9Z), and DLD (PDBid:2EQ9), (R) overlay of TGR (PDBid:5W1J) and MerA (PDBid:1ZK7), (S) overlay of Pdr (PDBid:1Q1W), TodA (PDBi­ d:3EF6), BphA4 (PDBid:4H4W), AIF (PDBid:5FS9), and MDHAR (PDBid:6KRT), (T) RdxR (PDBid:2V3A), (U) overlay of NPX (PDBid:1F8W), NOX (PDBid:5ER0), and CoADR (PDBid:4EQX), (V), Npsr (PDBid:6PFZ). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

oxidoreductases that do not naturally fall into the other four groups, but the interaction occurs on the re-face of FAD with AhpF interacting with can be characterized as a miscellaneous group containing additional both NAD(P)H and the disulfide on the re-face. The FPMOs have the segments/domains. Alkyl hydroperoxidase reductase subunit F (AhpF), lowest sequence identity to low Mr TrxR, and contain 40-200 additional which recycles peroxiredoxin AhpC, is the protein with the highest residues [43–47]. FMO has an additional helix-loop-helix motif, while sequence identity and DALI Z-score to the low Mr TrxR, but contains an NMO and BVMO have additional substrate-binding domains (Fig. 4L,M). additional flexible N-terminal domain consisting of two Trx-folds Fig. 5E shows that for NMO, both the NADPH and the substrate binds on (Table 1, Fig. 4K) [42]. Similar to the members of the previous group, the re-face of FAD. The two final enzymes in this group are

7 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Fig. 5. Active sites of selected flavoprotein reductases. The different structures are shown in cartoon and colored white. If a second monomer or a protein substrate is shown, it is colored in black. Cofactors and small molecule substrates are represented as sticks and colored by atom type. The carbon atoms are colored in a unique color for each enzyme. The active site of (A) high Mr TrxR (PDBid:3EAO and 2ZZC, NADPH from the latter), (B) low-Mr TrxR in FR form (PDBid:5VT3), (C) low Mr TrxR in FO form (PDBid:1TDE and 1TDF, NADPH from the latter), (D) GR (PDBid:1GRA and 1GRB, NAPDH from the latter), (E) NMO (PDBid:3S5W), (F) FNR (PDBid:3LZX), (G) NPX (PDBid:1F8W and 2NPX, NADH from latter), (H) CoADR (PDBid:4EQR), and (I) BphA4 (PDBid:2YVJ). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) heterodisulfide reductase (HdrA) and sulfide:quinone oxidoreductase 4.4. Oxidoreductases containing the low Mr TrxR-like fold and an (SQR), which both contain different additional domains, but do not additional C-terminal domain, but no Cys pair (4) utilize NAD(P)H in their reactions (Table 1, Fig. 4N,O) [48,49]. No redox-active cysteines are present in this group of enzymes, despite having a similar fold as seen for the previous group (Table 1, 4.3. Oxidoreductases containing the low Mr TrxR-like fold, an additional Fig. 4S). The overall structures of enzymes within this group are very C-terminal interface domain, and Cys pair(s) (3) similar, as can be seen from the structural overlay (Fig. 4S), all con­ taining an additional C-terminal domain (Fig. 4S,T). In contrast to the This group is part of the FDR family and contains different active Cys previous groups, the multimerization states vary [57]. For instance, the pair motifs (Table 1). The overall structures of the enzymes within this apoptosis-inducing factor (AIF), after accepting two electrons from group are very similar as can be seen from the structural overlay in NADH, undergoes a dimerization and conformational change [58–60]. Figs. 4P,Q,R. Structurally, in addition to the low Mr TrxR-like fold, these Rubredoxin reductase (RdxR) on the other hand, has a smaller C-ter­ enzymes contain a C-terminal interface/dimerization domain (Fig. 4P,Q, minal domain acting as a rubredoxin-binding domain [57]. Several en­ R) [12,50–54]. MerA and thioredoxin glutathione reductase (TGR) also zymes in this group have other proteins as redox partners/substrates. contain a fourth domain [55,56]. The substrates vary from disulfide The organization of the Fd reductase subunit of biphenyl dioxygenase proteins, such as Trx for high Mr TrxR, to sulfur-containing organic (BphA) is shown in Fig. 5I, displaying NADH on the re-face of the molecules, like glutathione disulfide (GSSG) for GR or inorganic ions isoalloxazine ring of FAD, and on the other side, the interaction with the 2+ such as Hg for MerA. Generally, the electrons flowfrom NAD(P)H on Fe2S2 cluster of Fd [61]. the re-face to the isoalloxazine ring of FAD, and further to the Cys pair on the si-face (Fig. 5A,D). In high Mr TrxR, the reduced Cys pair further 4.5. Oxidoreductases containing the low Mr TrxR-like fold, an additional reacts with another redox-active disulfide or selenosulfide pair in the C-terminal interface domain, and an active single Cys (5) C-terminal tail of the other monomer of the homodimer, which finally moves to react with Trx [30] (Figs. 5A and 2C). In GR, the disulfide on These enzymes have a similar structural arrangement as described the si-face reacts instead directly with GSSG [14] (Fig. 5D). The other for the two previous groups containing the low Mr TrxR-like fold and a C- enzymes of this group have a similar structural organization and elec­ terminal interface domain (Figs. 4U,V and Table 1). Persulfidereductase tron flow, but with small differences with respect to the substrate they (Npsr) has an additional rhodanese domain (Figs. 4V). What distin­ act upon. guishes the members of this group from the previous two groups, is the

8 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Fig. 6. TrxR-like FNRs involved in various redox pathways. For FNRs and FNR homologs, one protomer is colored, whereas the other protomer(s) are depicted in grey. (A) B. cereus FNR2 (PDBid:6GAS) as electron donor to Flds (PDBid:6GAQ) and NrdI (PDBid:2X2O), the latter acti­ vating the NrdF subunit (PDBid:4BMU) of bacterial class Ib RNR. (B) B. subtilis FNR YumC (PDBid:3LZX) can reduce Fd and Fld YkuN, the latter activating NOS (PDBid:1M7V). (C) C. tepidum FNR (PDBi­ d:3AB1) is reduced with electrons from the photosynthetic RC, via Fd. (D) S. aureus FNR IruO (PDBid:5TWB) is shown to reduce heme-degrading IsdG (PDBid:2ZDO) and IsdI (PDBid:2ZDP), as well as hydroxamate-type siderophores. (E) Tetrameric B. cereus and S. aureus Bdrs (PDBid:7A76, 7A7B, 7APR) reduce the LMW thiol bacillithiol disulfide (BSSB) to bacillithiol (BSH). *closest structural ho­ molog: B. thermoproteolyticus Fd (PDBi­ § d:1IQZ), closest structural homolog: ¶ B. cereus Fld2 (PDBid:6GAQ), closest structural homolog: P. aeruginosa Fd (PDBid:2FGO). Arrows indicate the direc­ tion of electron flow.

active site single redox-active Cys found on the si-face of the isoalloxa­ 5. TrxR-like NAD(P)H-dependent cystine-independent FAD- zine ring of FAD (Fig. 5G and H). In NPX and NADH oxidase (NOX), containing oxidoreductases reducing hydrogen peroxide or dioxygen to water, the reaction propa­ gates through the generation of an active site cysteine sulfenic acid [12, Many bacteria contain proteins originally annotated as TrxRs, due to 62–64] (Fig. 5G). Coenzyme A disulfide reductase (CoADR) also con­ the high sequence similarity with the low Mr TrxRs (or generally an­ tains a single Cys on the si-face, but here, coenzyme A disulfide notated as NAD(P)/FAD-dependent oxidoreductases). However, many (CoA-S-S-CoA) reacts directly with the cysteine generating a of these do not contain the conserved CXXC motif of low Mr TrxRs, and CoA-S-S-Cys intermediate, before a full reduction by NAD(P)H through therefore most likely do not function as TrxRs in the reduction of the flavin [65] (Fig. 5H). disulfide-containing substrates. Several of these bacteria lack an anno­ In the remaining part of this review, selected oxidoreductases con­ tated FNR, nor encode proteins with sequence similarity to the plant- taining only the low Mr TrxR-like fold (structural group (1)) will be type FNRs that are found in many bacteria. Therefore, it has been discussed in more detail. reasonable to suspect that certain oxidoreductases annotated as TrxRs in

9 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Fig. 7. Conformational variation in TrxR- like FNRs. The structures show variations in domain conformation due to different rota­ tions of the NADPH-domains relative to the FAD-domains. Only monomers are depicted in Figures A–D. (A) Overlay of B. cereus FNR2 (PDBid:6GAS) and B. subtilis FNR YumC (PDBid:3LZX) (NADPH from the latter), (B) T. thermophilus FNR (PDBid:2ZBW), (C) C. tepidum FNR (PDBi­ d:3AB1), and (D) overlay of S. aureus IruO (PDBid:5TWB) and B. cereus FNR1 (PDBid:6GAR). In (E), the open FNR conformation is shown, allowing Fld binding [41] (calculated with the ClusPro2.0 server [144]) (PDBid:6GAS and 6GAQ), whereas in (F), the closed, preferred hydride transfer conformation of FNR is shown (PDBid: 6GAR).

+ selected bacterial species may in fact function as FNRs in these bacteria, bound (PDBid:3LZW, 3LZX) [70], NADP is bound more than 15 Å away especially since they also encode Flds. Even though the firstaccount of a from the FAD group, too far away for productive hydride transfer [72]. A TrxR-like FNR was published nearly 20 years ago, the TrxR annotation nearly identical orientation is seen in B. cereus FNR2 (PDBid:6GAS) [41] remains in many genomes. Moreover, some bacteria encode multiple (sequence identity 78%), and a similar but slightly different orientation homologous variants of potential FNRs, further suggesting that some can is seen in Thermus thermophilus FNR (PDBid:2ZBW). However, other FNR function specifically as FNRs in reducing Fds or Flds, whereas other crystal structures reveal different orientations of the NAD(P)H domains FNRs may serve different catalytic functions. During the last decade, relative to the FAD domains, as seen in the crystal structures of B. cereus different functions have been suggested for this type of TrxR-like NAD FNR1 (PDBid:6GAR) and S. aureus FNR (IruO, iron utilizing oxidore­ (P)H-dependent cystine-independent FAD-containing oxidoreductases. ductase) (PDBid:5TWB, 5TWC) [73] (Fig. 7A–D). Thus, a domain rota­ An overview of functions and redox pathways assigned for the selected tion is highly likely to take place in order to position the NAD(P)H close TrxR-like FNRs discussed below is presented in Fig. 6. Three types of enough to reduce the FAD cofactor, and for allowing substrate binding. such oxidoreductases will be discussed in detail in Sections 5.1-5.3 This notion is further strengthened through studies by Muraki and below. The overview in Fig. 6 demonstrates the variations in the co-workers, who solved the crystal structure of C. tepidum FNR (PDBi­ orientation of the FAD-binding domains relative to the NAD(P)H- d:3AB1) [23]. This structure revealed a unique homodimer in which the binding domains of the different enzymes, and gives an overview of domains are arranged asymmetrically, with one NAD(P)H domain pre­ the redox partners/substrates identified for these enzymes in Bacillus sent in an open conformation, and the other with the NAD(P)H domain cereus, Bacillus subtilis, Chlorobaculum tepidum, and Staphylococcus in a closed conformation. The same study also demonstrated decreased aureus. FNR activity when replacing conserved glycine residues with prolines in the hinge region, again suggesting the importance of a domain rotation for catalysis, and that the hinge-motion is involved in binding of the 5.1. Ferredoxin/flavodoxin NADP+ reductase (FNR) pyridine nucleotide, as well as Fd or Fld in FNRs. An interesting model organism for investigating TrxR-like FNRs has The firstTrxR-like FNRs were isolated from C. tepidum in 2002 [66], been the Firmicute B. cereus. B. cereus strain ATCC 14579 contains three and since then, several homologs have been found in e.g. Firmicutes [41, genes encoding TrxR-like FNRs; BC0385 (FNR1), BC4926 (FNR2), and 67,68], α-proteobacteria [19], and thermophilic prokaryotes [24,69]. As BC1495 (FNR3, recently shown to function as a bacillithiol disulfide described above, TrxR-like FNRs, despite their structural similarity with reductase (Bdr) (see Section 5.3)). Although these were originally an­ low Mr TrxRs, cannot function as TrxRs due to the lack of a catalytic notated as TrxRs, none contain the TrxR catalytic CXXC motif. A mul­ CXXC-motif. Similar to the TrxRs, the TrxR-like FNRs are homodimeric. tiple sequence alignment showed that the TrxR-like FNRs cluster in three This is in contrast to other types of FNRs, which, with a few exceptions, different branches on a phylogenetic tree, however, FNR1 and FNR2 are monomeric [19]. Each TrxR-like FNR protomer has the two domains, share a higher sequence identity than they do with Bdr [68]. B. cereus with Rossmann-like three-layer ββα sandwich nucleotide binding folds also contains two flavodoxins, Fld1 and Fld2, as well as the for both FAD and NAD(P)H. The NAD(P)H-binding domain is inserted flavodoxin-likeprotein NrdI. Thus, the B. cereus FNR/Fld redox network between two sections of the FAD-binding domain, connecting to the has been a good starting point to compare and contrast the structure and FAD-binding domain by a hinge region consisting of an antiparallel function of different FNRs and Flds, as well as the FNR-Fld interaction β-sheet. The two domains can rotate relative to each other, as suggested and electron transfer. by the crystal structures of TrxR-like FNRs that have crystallized in The crystal structure of B. cereus FNR2 [41], as also seen from the several different conformations [23,41,70] (Fig. 7A–D). This domain structure of B. subtilis YumC FNR [70], show that the NAD(P)H-binding rotation is unique from other types of FNRs, and different from the domain adopts a conformation that would disallow hydride transfer domain rotation enabling catalysis in low Mr TrxR [33,36,71]. However, from the NAD(P)H cofactor to the FAD cofactor. Here, the NAD(P) a rotation between the FAD- and NAD(P)H-binding domains is likely to H-FAD distance is 16.9 Å (flavin N5 to NAD(P)H C4), demonstrating play a role in the TrxR-like FNR catalytic mechanism as well. In the that a large-scale conformational rearrangement must take place for + crystal structure of a B. subtilis TrxR-like FNR (YumC) with NADP

10 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826 hydride transfer. However, this open conformation appears to give more room for substrate binding (Fig. 7A,E). In the open FNR2 conformation, the substrate Fld has been suggested to dock close to the FAD cofactor [41], and hence, suggesting that electron transfer must be favored in the open conformation as seen in these crystal structures. To the contrary, B. cereus FNR1 has been crystallized with the NAD ◦ (P)H-binding domain rotated 60 relative to the NAD(P)H-binding domain seen in the structure of B. cereus FNR2 [41] (Fig. 7D,F). This rotation has implications for both substrate binding and the hydri­ de/electron transfer between NAD(P)H and FAD in FNR, and to FMN in the Fld substrate, respectively. In FNR1, the NAD(P)H-FAD distance would be 10.2 Å (flavin N5 to NAD(P)H C4) [41]. The closed confor­ mation seen in this structure could allow for electron transfer in the FNR1 conformation, suggesting that the conformation of FNR1 is closer to the preferred hydride transfer conformation. Furthermore, attempts of docking Flds to the B. cereus FNR1 structure gives no single consensus , and only sites distant to the FAD cofactor [41]. Thus, the computed potential Fld binding site would allow for electron transfer in the FNR2 conformation, but not in the FNR1 conformation, further supporting the hypothesis that substrate binding occurs in the open conformation of FNR2 (Fig. 7E and F). In addition to the structural aspects giving insight into FNR domain rotation and substrate binding, as demonstrated by the B. cereus FNR1 and FNR2 crystal structures, interesting findingsrelated to function have been made based on the B. cereus FNR/Fld redox network. Ribonucle­ otide reductases (RNRs) catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides [74–76]. In order for the re­ action to take place in the manganese-bound class Ib RNR, formation of III • a Mn 2-tyrosyl radical (Y ) cofactor in the NrdF subunit is assisted by the flavodoxin-like protein NrdI, which reacts with oxygen to produce an oxidant used in the activation of the manganese cluster [77,78]. As NrdI has been shown to be involved in this pathway for more than a Fig. 8. Proposed IruO mechanism for the reduction of Fe(III)-side­ decade [77,79], it was only in 2015 that a study linked the role of a rophores. The reduced IruO dimer (PDBid:5TWB) mediates electron transfer TrxR-like FNR in Lactococcus lactis to ribonucleotide reduction [80], and from NADPH, via a FADsq intermediate, for the reduction of Fe(III)- desferrioxamine B and Fe(III)-ferrichrome A. a native NrdI reductase was first described in a study by Hersleth and co-workers a year later, identifying a missing piece of the class Ib RNR redox pathway [68]. The latter study showed similar dissociation con­ FAD re-face (including e.g. B. cereus FNR1) [41]. Mutational studies of stants for the B. cereus FNR(1/2/3)-NrdI redox pairs, however, the FAD-stacking aromatic residue have suggested a shielding role of steady-state kinetics studies showed that the turnover number of FNR2 FAD from exposure to solvent during catalysis [23,70], however, none of was >10-fold and >30-fold higher than the turnover numbers of FNR1 these studies have mutated the aromatic residue to a hydrophobic, and FNR3, respectively, in the reduction of NrdI. The study also aliphatic residue such as Val. Further studies are required to determine confirmed that the FNR2/NrdI redox system activated NrdF in vitro, whether the interchange of the aromatic residue to Val affects the • generating the highest Y yield, as compared to the FNR1-NrdI and function of the TrxR-like FNRs. Another difference between B. cereus FNR3-NrdI pairs. Thus, FNR2 was established as an endogenous redox FNR1 and FNR2 is that FNR1 has a much longer C-terminal helix than partner involved in the activation of NrdI, and hence, class Ib RNR in FNR2 [41]. The role of the C-terminal helix in TrxR-like FNRs is still B. cereus (Fig. 6A). Furthermore, the same group found that FNR2 re­ unclear, however, functions involved in substrate binding and/or elec­ duces B. cereus Fld1 and Fld2 at significantly higher rates than the two tron transfer [82], and hydride transfer [83] have been proposed. other FNRs, with FNR2-Fld2 being the most efficient electron transfer Another example illustrating substrate diversity and lack of speci­ pair (>200-fold higher turnover than FNR1-Fld2 and >3000-fold higher ficityamong FNRs, as often seen for electron transfer complexes [84], is than FNR3-Fld2), with a similar reduction rate to that of the E. coli the B. subtilis YumC FNR. This FNR was originally characterized as a plant-type FNR-Fld pair [81]. Also, the FNR2-Fld2 pair showed a redox partner to iron-sulfur Fds, showing a remarkably higher reactivity >90-fold higher turnover rate than the electron transfer pair FNR2-NrdI towards Fe4S4 type Fd than those for Fe2S2 type Fd reduction [19], in [41]. Insight into possible reasons for the differences in electron transfer contrast to other types of FNRs whose physiological electron donor/­ rates and substrate specificities of FNR1 and FNR2 became apparent acceptors are considered to be Fe2S2 type Fds [85]. Later on, YumC FNR upon inspection of their crystal structures. The conserved C-terminal was also confirmedto function as a native redox partner to the Fld YkuN subdomain of one monomer stretches over the FAD cofactor in the other in B. subtilis [86], the latter demonstrated to function as a redox partner monomer [23], as also seen from most published structures of TrxR-like involved in nitric oxide synthase (NOS) activity [21] (Fig. 6B). More­ over, another interesting aspect of FNRs is seen for the C. tepidum FNR, FNRs. Three residues in this helix, an aromatic residue (His, Tyr, or Phe) + followed by two aliphatic hydroxyl-containing residues (Ser or Thr) which catalyzes the reduction of NAD(P) to NAD(P)H with reduced Fd, stabilize the FAD cofactor by π-π interactions and hydrogen-bonding the latter accepting electrons from the photosynthetic reaction center [23,41,70]. Although these residues were suggested to be a common (RC) of green sulfur bacteria during the finalstep of the photosynthetic feature in all TrxR-like FNRs, it was recently shown that the aromatic electron transport chain [23,85,87] (Fig. 6C). Whether either of the residue is replaced by a conserved Val in several FNRs, proposing a di­ B. cereus FNRs may function as redox partners to the two Fds encoded in vision into two phylogenetic subclasses of TrxR-like FNRs; one where an the B. cereus genome remains to be investigated. aromatic residue is stacked opposite to the FAD on the FAD re-face Despite the lack of substrate specificity, as well as direction of the (including e.g. B. cereus FNR2), and one where Val is positioned on the electron flow in the above described FNRs, we define these FNRs as

11 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Fig. 9. The bacillithiol disulfide reduc­ tase Bdr. (A) Structure showing the S. aureus Bdr monomer (PDBid:7A7B and 7APR. Biological homotetramer depicted in Fig. 6E), displaying various structural fea­ tures: NADPH and FAD cofactors (sticks, colored by atom type), Tyr128, Phe51, and loop (involved in gating, colored teal), Cys14 (colored teal), and putative BSSB binding channel (pink surface). (B) Model of BSSB substrate. (C) Proposed Bdr reaction mechanism for the reduction of BSSB to 2 x BSH. NADPH binds and reduces FAD, + NADP leaves and BSSB binds, and BSSB is reduced to 2 x BSH through a thiol-thiolate pair FAD C4a-cysteine adduct intermediate. (For interpretation of the references to color in this figurelegend, the reader is referred to the Web version of this article.)

“classical FNRs”, in respect to redox networks involving Flds/Fds. siderophores, staphyloferrin A and B [108]. In addition, S. aureus can Recently, other and completely different functions have been described internalize iron-bound siderophores produced by other bacteria and for FNRs and FNR homologs in the literature, displaying an intriguing fungi through the use of membrane anchored transporters; the SstABCD evolutionary divergence among these structurally related enzymes. For uptake system for catecholate-type siderophores [109], and the instance, although FNR1 from B. cereus is able to reduce Flds and NrdI at FhuBGC2-D2/D1 uptake system for hydroxamate-type siderophores lower catalytic efficiencies [41,68], an FNR1 homolog from S. aureus, [110,111]. Iron release from the internalized Fe(III)-siderophore com­ the TrxR-like FNR IruO, has been reported to serve different functions. plexes requires a one-electron reduction of the Fe(III) complex to ferrous IruO functions in the reduction of heme degrading enzymes, IsdG and iron (Fe(II)) [112]. The iron-regulated nitroreductase NtrA has been IsdI [88,89], as well as in the reduction of hydroxamate-type side­ shown to be essential for the utilization of the polycarboxylate-type rophores [73] (Fig. 6D). Moreover, B. cereus FNR3 (Bdr), a poor redox siderophore Fe(III)-staphyloferrin A [113]. IruO, on the other hand, partner to Flds and NrdI, has recently been shown to serve yet another has been demonstrated to reduce the hydroxamate-type siderophores Fe function, namely as a reductase of oxidized bacillithiol [90], as initially (III)-desferrioxamine B and Fe(III)-ferrichrome A (Figs. 6D and 8) [73, reported for Bdr from S. aureus (sequence identity 60%) [10,91] 113]. Based on UV–vis spectroscopic investigations, Murphy and co­ (Fig. 6E). IruO and Bdr will be discussed in sections 5.2 and 5.3, workers have proposed that each FADox cofactor in the IruO dimer ac­ respectively. cepts one electron each from NAD(P)H, where one electron is transferred across the interacting FAD-binding domains in the adjacent subunits, resulting in the FAD state in both protomers (1 NAD(P)H to 1 5.2. Iron utilizing oxidoreductase (IruO) sq IruO dimer). Ultimately, is was shown that the FADsq form was able to reduce the Fe(III)-siderophore complex, indicating that reduced IruO S. aureus does not have any genes coding for Flds. It does encode, mediates the transfer of electrons from NAD(P)H, via a FAD interme­ however, a TrxR-like FNR, IruO, which is a close homolog of the B. cereus sq diate, to reduce two Fe(III)-siderophore complexes [73] (Fig. 8). Proof of FNR1 (sequence identity 45%). As S. aureus also encodes an Fd, it would this unprecedented two single-electron transfer mechanism from be reasonable to assume that IruO functions as a redox partner to Fd NADPH is lacking. NADPH is known to be a mandatory 2-electron instead, however, to our knowledge, such activity has not been reported. (hydride) donor, so a possibility is first to generate FAD/FADH2 and If the FNR1 homolog preferentially reduces Fd in B. cereus remains to be • then 2FADH , but a stepwise hydride transfer has recently been shown investigated, however, for S. aureus IruO, other interesting redox part­ to be possible in biological systems [114]. ners and roles in iron acquisition have been discovered. As also seen in the B. cereus FNR1 structure, structural analysis of S. aureus is a widely studied human pathogen and a leading cause of IruO (S. aureus NWMN0732) revealed that a conserved valine residue global mortality, causing a number of diseases [92,93]. Iron acquisition (Val324) is positioned on the FAD re-face, stabilizing the isoalloxazine is required for bacterial metabolism, growth, and the ability to sustain a ring, placing B. cereus FNR1 and S. aureus IruO in the same phylogenetic successful infection by this bacterium, and obtaining iron is critical for FNR clade [41,73]. The crystal structure of reduced IruO (PDBid:5TWB) these purposes as the concentration of freely available iron in (Cys248 and Cys265 in hinge region and FAD-binding domain, respec­ mammalian hosts is negligible [94,95]. Consequently, S. aureus has tively) has revealed that this form is the preferred substrate-binding evolved different strategies for iron acquisition. One such strategy is form, with a putative siderophore binding site in a pocket located next through heme degradation, regulated by the iron-regulated surface to the re-face of the FAD isoalloxazine ring. In the structure of oxidized determinant (Isd) system [96]. Host heme-containing proteins hemo­ ◦ IruO, (PDBid:5TWC), the NAD(P)H-domain is rotated 90 relative to the globin and haptoglobin are bound by wall-anchored proteins (IsdA, FAD domain, with the formation of an intramolecular disulfide bond IsdB, IsdC, and IsdH), which extract heme, and shuttle it to the bacterial between Cys248 and Cys265. This rotation results in a conformational membrane [97–103]. Once transported across the membrane into the change in the suggested siderophore binding site, rendering it signifi­ cytoplasm by an ABC transporter (IsdE, IsdF, and possibly IsdD) [98, cantly narrower. However, the general significance of this observation 104,105], the degradation of the porphyrin ring of heme, and release of for IruO can be questioned, as this cysteine pair is not conserved among iron, is catalyzed by the paralogous heme degrading monooxygenases other Staphylococcus species, nor other Firmicutes. Based on these IsdG and IsdI [106,107]. IruO has been shown to supply electrons to findings, together with a higher Fe(III)-siderophore reductase activity IsdG and IsdI (Fig. 6D), and has been proposed to be the biological seen for reduced IruO, it has been suggested that the latter is the reductase associated with these enzymes in heme degradation in preferred catalytic form [73]. Murphy and co-workers show that IruO S. aureus [89]. prefers NADPH over NADH as a source of electrons. However, in Another iron acquisition strategy applied by S. aureus, is scavenging contrast to FNR2 and FNR YumC, IruO and FNR1 do not contain the of Fe(III) bound to host proteins using two endogenously synthesized

12 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

′ common HRRXXXR sequence motif for binding of the 2 P phosphate structural similarity to other members of the tDBDF superfamily group of NADPH. Alignment of the FNR YumC and FNR1 structures (Figs. 6E and 9A) [90]. It has been shown that Bdr can utilize both NADH indicates the presence of Arg and Lys residues in the vicinity of the and NADPH [91], but the structure of S. aureus Bdr expressed in E. coli ′ potential binding site for 2 P phosphate of NADPH in IruO, although no contained only naturally bound NADPH. Bdr does not contain the ′ IruO structure with bound NADPH/NADH currently exists. common HRRXXXR sequence motif for binding of the 2 phosphate group Interestingly, a recent paper reported TrxR-activity in the reduction of NADPH, but the phosphate is stabilized by an Arg and a Lys residue. A of Trx1 and Trx2 for the FNR/IruO from another S. aureus strain key question is how this enzyme is able to reduce BSSB without the use (NCTC8325) (sequence identity 100%). The authors named it TrxR2, as of a redox-active Cys pair. The Rossmann-like FAD-binding domain of it showed higher substrate specificity and catalytic efficiency towards Bdr contains the canonical glycine-rich signature sequence motif Trxs [115] than reported for IruO towards siderophores or IsdG/I [73, GXGXXG (GGGPC14G in S. aureus and B. cereus). The single Cys residue 89]. The crystal structure of TrxR2 (PDBid:6A4J) has an identical of this motif is conserved in Bdr homologs in Firmicutes, however, it is structural disposition to that of oxidized IruO (PDBid:5TWC) [73], with replaced by other conserved residues within Bdrs from other phyla the two cysteines (Cys248 and Cys265) forming a disulfide bridge. The suggested to use BSH or N-methyl-bacillithiol [90]. Antelmann and co­ study reports that a mixed-disulfidecomplex can be formed between the workers showed that this Cys is important for reduction of BSSB by Trx substrate and the TrxR2 C-terminal redox-active cysteine, support­ S. aureus Bdr, based on the inactive Bdr C14A variant [10]. The Bdr ing that a redox reaction following the pathway described for TrxRs can crystal structures have revealed, however, an unlikely participation of take place. An intriguing feature of oxidized IruO/TrxR2 is that the Cys in the reduction of the BSSB substrate, due to the buried location of conserved active site CXXC motif in the pyridine-nucleotide-binding the Cys, ~8 Å away from the FAD isoalloxazine ring, and the conserved domain is missing, placing the two above-mentioned cysteine residues orientation of an α-helix closely lining and shielding the GGGPCG motif. in a hinge region between the cofactor-binding domains. This location Moreover, the structures show inadequate space for BSSB entry or does not place the redox-active disulfide close to the isoallozaxine ring binding in the proximity of the Cys, as well as in the space between Cys of FAD (re-face), crucial for reduction of the disulfide by the reduced and the FAD cofactor (Fig. 9A). flavin, as seen in the TrxR FO conformation [36]. Also, as mentioned A probable BSSB binding site directly above the isoalloxazine ring above, this Cys-pair is not conserved among homologous proteins in has been proposed, possibly regulated through an amino acid gating other Firmicutes, so the potential significance has to apply only for mechanism of the channel on the re-face of the isoalloxazine ring (gated certain S. aureus strains. Future studies to elucidate the detailed struc­ by Phe, Tyr, and a loop movement) (Fig. 9A and B). Based on the latter tural basis of IruO/TrxR2 activation are needed to resolve its proposed findings, a simpler and more direct reaction mechanism has been pro­ unusual TrxR-activity. posed for Bdr, resembling the classical flavoprotein disulfide reductase If the homologous S. aureus IruO/TrxR2 and B. cereus FNR1 can mechanism as described for TrxR [38,126] (Fig. 3B and Section 3), but function as redox partners to Fd remains to be elucidated. Regardless, without the cysteine-based dithiol step (Fig. 9C) [90]. the diversity in substrate specificity reported for these types of FNRs illustrates an interesting evolutionary path where nearly identical 6. TrxR-like NAD(P)H-independent cysteine-dependent FAD- structurally homologous enzymes have adapted specialized roles in containing oxidoreductases distinct cellular processes. The functional diversity among structurally related flavoprotein 5.3. Bacillithiol disulfide reductase (Bdr) oxidoreductases is vast. Also, an increasing number of proteins of the TrxR superfamily, structurally similar to low Mr TrxR, have been The TrxR- and FNR-like protein Bdr (previously named YpdA) described, containing interesting and atypical features. Although these functions as an essential NAD(P)H-dependent reductase of oxidized flavoenzymesare composed solely of the TrxR-like fold (placing them in bacillithiol disulfide (BSSB). Bacillithiol (BSH) is a LMW thiol used by oxidoreductase group (1)) including the two canonical ββα-sandwich low G+C Gram-positive Firmicutes, such as S. aureus and B. cereus, as a Rossmann-like fold domains with FAD bound in one domain and the defense mechanism to buffer the intracellular redox environment and redox-active cysteine-containing motif located in the other domain, counteract oxidative stress. The LMW thiol GSH is the most abundant several such atypical TrxR-like oxidoreductases have been shown to thiol antioxidant contributing to the control of redox homoeostasis, and function independently of NAD(P)H as the physiological electron donor is produced in most eukaryotes, Gram-negative bacteria, and some [127–132]. This is largely due to the fact that these protein sequences Gram-positive bacteria. In redox reactions, GSH is continuously oxidized lack the conserved sequence motifs GXGXXG/A and the often common to GSSG, which is recycled back to GSH by GR [116], to maintain the HRRXXXR motif necessary for pyridine nucleotide binding, as seen in required GSH/GSSG ratio important for the cellular redox balance. canonical TrxR and many other TrxR-like oxidoreductases, however, the Furthermore, an important post-translational modification for regu­ presence of this arginine-rich motif is not an essential feature in several lating protein function and protecting exposed cysteine residues from NAD(P)H-utilizing oxidoreductases either. Hence, the NAD(P)H-binding irreversible oxidative damage is the reversible formation of GS-S-protein domain of previously described oxidoreductases in this review has for disulfides(S-glutathionylation) on proteins [116]. Analogously to GSH, the NAD(P)H-independent oxidoreductases been termed pseudo-NAD(P) low G+C Gram-positive Firmicutes instead utilize BSH for these func­ H-binding domain, or alternatively redox-active disulfide domain, in tions, as cofactors of enzymes in scavenging of e.g. reactive oxygen these divergent flavoenzymes. Structural investigations have revealed species (ROS), detoxification of toxins and antibiotics, protection that these NAD(P)H-independent oxidoreductases and low Mr TrxR against heavy metals and in metal storage [117,118], and protection of share a similar homodimeric domain architecture, however, may differ protein thiols through S-bacillithiolation [119–125]. in the domain organization and interactions, as their crystal structures De-bacillithiolation of proteins is catalyzed by bacilliredoxins (Brxs). have conformations differing from the FO and FR forms observed in low The resulting Brx-SSB intermediate is reduced by BSH, leading to Mr TrxR structures (Fig. 4F–J versus A-B). + oxidized BSSB, which in turn is recycled to BSH by the FAD-containing For instance, NAD(P) -independent flavin dithiol oxidases contain NAD(P)H-dependent disulfide oxidoreductase Bdr [10]. the TrxR-like fold, but catalyze the formation of disulfide bridges in The recently solved crystal structures of Bdr homologs from S. aureus small molecules through the redox-active CXXC motif and FAD in a NAD + and B. cereus (the latter previously referred to as FNR3) (PDBid:7A76, (P) -independent manner. Despite having variations in their substrate 7A7B, 7APR) revealed a unique homotetrameric assembly not described specificity,this group of enzymes share a common reaction mechanism + for any other TrxR or FNR oxidoreductases (Fig. 6E) with each protomer using molecular oxygen instead of NAD(P) as a cosubstrate and ter­ containing the NAD(P)H and FAD-binding domains, and with high minal electron acceptor for the oxidation of dithiols to disulfides in

13 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Fig. 10. FFTR. (A) Crystal structure of homodimeric C. acetobutylicum FFTR in complex with Trx (PDBid:6GND). (B) Model of the FFTR-Fd complex, where a C. acetobutylicum Fd model has been docked to each FFTR monomer (PDBid:6GNA) at the interface of the FAD-binding and pseudo-NAD(P)H-bind­ ing domains using the ClusPro2.0 server [144]. The C. acetobutylicum Fd ho­ mology model was generated with the SWISS-MODEL server [145] using the closest structural homolog of Fd from Clostridium pasteurianum (PDBid:1CLF) similar to Buey et al. [131]. Redox centers and cofactors are shown as sticks and colored by atom type. The FAD-binding domains and pseudo-NAD(P)H-binding domains are colored light grey and dark grey, respectively, whereas Fd and Trx are colored pink and yellow, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) natural products with antitumor, antibiotic, and virulence factor activ­ Fig. 11. DDOR. (A) Crystal structure of homodimeric G. violaceus DDOR ities [127,128,133,134]. DepH catalyzes the disulfidebond formation in (PDBid:5N0J), with each monomer colored grey and black, respectively. The the anticancer prodrug romidepsin. Characterized from Chromobacte­ juxtaposed FAD cofactors (FAD1 and FAD2), as well as the redox-active disul­ rium violaceum, DepH (PDBid:4JN9, 4NTE) (Fig. 4F) is structurally fide are shown as sticks, and colored by atom type. In the grey protomer (left), related to TrxR, containing the classical Rossmann-like FAD-binding the pseudo-NADPH domain is depicted in darker grey. (B) Schematic repre­ + domain and, unlike TrxR, a pseudo-NAD(P) -binding domain. In sentation of the DDOR reaction mechanism, involving a proposed sulfhydryl contrast to TrxR, DepH contains a loop which physically impedes the external electron donor. + binding of NAD(P) , and lacks the motifs necessary for NAD(P) H-binding [127,128]. Other members of this group of structurally and the newly characterized isolated TrxR (TRi) (PDBid:5NII) (Fig. 4G), related, specialized oxidoreductases include GliT (PDBid:4NTC) [127] described from Desulfovibrio vulgaris [130], which both reduce Trx in a (Fig. 4F), catalyzing the disulfidebond formation in the finalstep of the NAD(P)H-independent manner. In DTR, a C-terminal extension with an biosynthesis of the virulence factor gliotoxin in the fungus Aspergillus aromatic residue stacking over the FAD cofactor is present in the fumigatus [127,134], and HlmI (PDBid:4NTD) [127] (Fig. 4F), involved structure, not found in canonical TrxR but resembling the C-terminal in the biosynthesis of the antibiotic holomycin in Streptomyces clav­ subdomain in certain FNRs, and has been proposed to participate in a uligerus [127,133]. The two latter oxidoreductases are, as also seen from regulatory mechanism in DTR. Till date, no physiological donors have the DepH structure, composed of the TrxR-like fold, however, the been fully established for DTR or TRi, however, Fld has been suggested FAD-binding domain of Hlml contains an extended C-terminal α-helix as a candidate for the latter. The structure of another flavoprotein ho­ [127]. mologous to low Mr TrxR has recently been solved from Clostridium Other intriguing examples of expanded structural and mechanistic acetobutylicum. As for DTR and TRi, this unique oxidoreductase also repertoires of TrxR-like oxidoreductases, which are involved in thiol- functions as a redox partner to Trx in a NAD(P)H-independent manner, based redox regulation and the reduction of Trx have also been however, Trx reduction has been shown to be driven by Fd, and this described. These include e.g “deeply-rooted TrxR” (DTR) (PDBid:5J60) enzyme has therefore been named ferredoxin-dependent flavin thio­ (Fig. 4G), which is found in some cyanobacteria and marine algae [129], redoxin reductase (FFTR) [131] (Fig. 4H). FFTR shows high structural

14 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

Fig. 12. AclR structure and mechanism. (A) Crystal structure of homodimeric A. oryzae AclR (PDBid:7BR0), with each monomer colored grey and black, respectively. The FAD cofactor and the redox-active CXXH motif are shown as sticks, and colored by atom type. In the grey protomer (left), the pseudo-NADPH domain is depicted in darker grey. (B) The active site of AclR, with the FAD cofactor and the redox-active noncanonical CXXH motif as sticks, colored by atom type. For com­ parison, the AclR active site is overlayed with the + active site of the homologous NAD(P) -indepen­ dent flavin dithiol oxidase GliT from A. fumigatus (PDBid:4NTC), displaying the canonical CXXC motif. The latter structure is displayed in pale green. (C) AclR mechanism involving spiro-ring formation and C–S bond formation of a precursor in the biosynthesis pathway of aspirochlorine. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) similarity to canonical TrxR, and hence, differs structurally from the form of FFTR, allowing for interaction with the substrate. In the FFTR functionally related ferredoxin-thioredoxin reductase (FTR) [135]. crystal structures, the isoalloxazine ring of FAD remains Another example of a structurally unique NAD(P)H-independent solvent-exposed. It is proposed that the modes of flexibility, associated TrxR-like oxidoreductase is diflavin-linked disulfide oxidoreductase with the flexible loop region and hinge-like opening and closing, may (DDOR) [132] (Fig. 4I). DDOR contains an exceptional structural feature further allow for different conformations of FFTR, altering the position of two FAD molecules bound in each subunit. of the redox-active cysteines during the catalytic cycle, triggering the A newly published paper by Hertweck and coworkers has presented switching from the FR to the FO conformation. This conformational structural and functional findings on yet a unique TrxR-like oxidore­ switch could also be initiated by the reduction of FAD and release of the ductase, AclR, from the fungus Aspergillus oryzae [136] (Fig. 4J). This binding-partner Fd. Fd, the reducing agent of FFTR, is assumed to bind novel enzyme has been shown to catalyze both carbon-sulfur bond to FFTR in the FR state, suitable for electron transfer from Fd to FAD migration and spiro-ring formation in the biosynthesis pathway of [131]. A putative binding-site for Fd, identifiedas an electron donor of aspirochlorine, an antifungal and toxic cyclodipeptide. AclR functions in FFTR, has been investigated through molecular docking (of Fd by Clu­ an NAD(P)H-independent manner, contains a noncanonical CXXH motif sPro2.0 server, resulting in similar orientation as suggested by Balsera crucial for catalysis, and binds a FAD cofactor suggested to act as a and coworkers [131]), revealing a probable Fd binding-pocket located at structural stabilizer and not as a redox-active cofactor during catalysis. the interface of the two domains of FFTR, placing the Fe–S cluster of Fd FFTR, DDOR, and AclR will be further discussed in Sections 6.1 - 6.3, close to the FAD cofactor of FFTR (Fig. 10B). The conformation observed respectively. in the FFTR structures, governed by the extended loop-region and allowing for binding of Fd, would be restricted in TrxR where the two β 6.1. Ferredoxin-dependent flavin thioredoxin reductase (FFTR) domains are connected through the less flexible -strand linker.

The first crystal structures of two FFTR paralogues from 6.2. Diflavin-linked disulfide oxidoreductase (DDOR) C. acetobutylicum, FFTR1 and FFTR2 (PDBid:6GNC, 6GNA, 6GNB), were recently solved by Balsera and coworkers [131], 35 years after the first Another study has recently described an additional and fascinating discovery of a flavoproteinshown to function with Fd and not NADPH in example of unprecedented TrxR-like oxidoreductases, DDOR, found the reduction of Trx in Clostridium pasteurianum [137]. FFTRs are found exclusively in oxygenic photosynthetic prokaryotes. The crystal struc­ almost exclusively in Firmicutes, with the exception of Bacillus species, tures of DDORs from Gleobacter violaceus and Synechocystis sp. have been and widely distributed in Clostridia [131]. In certain clostridial strains, solved (PDBid:5N0J, 5ODE, 5K0A) [132]. The perhaps most intriguing the Trx system solely relies on FFTR. Together with DTR and TRi, FFTR structural feature of DDOR, as the name implies, is the presence of two is structurally homologous to low Mr TrxR, and according to amino acid juxtaposed FAD molecules in each monomer of the DDOR homodimer sequence and structure conservation, DTR, TRi, FFTR, and low Mr TrxR (Fig. 11). One of the flavins,FAD1, is located in the FAD-binding domain share a common evolutionary ancestor. Homodimeric FFTR shares a analogously to what is seen for oxidized FAD in TrxRs. A second FAD, common overall structural architecture with low Mr TrxR with each FAD2, is located at the interface between the FAD-binding and pseu­ monomer being composed of two Rossmann-like fold domains. In the do-NAD(P)H-binding domains, with the isoalloxazine ring stabilized by FAD-binding domain, an aromatic residue stacks with the FAD isoal­ aromatic residues forming π-π stacking interactions. The orientation of loxazine ring on the si-face, forming π-π stacking interactions. This ar­ the juxtaposed FAD1 and FAD2 isoalloxazine rings suggests a possible omatic residue is part of a conserved motif, the specificity-determining direct interflavin redox transfer. The simultaneous binding of two FAD positions (SDPs), unique in FFTRs. The redox-active disulfideis located cofactors, and the presence of a C-terminal extension, indicates that in the pseudo-NAD(P)H-binding domain, analogously to what is seen in conformational flexibility and domain-rotation in DDORs is severely TrxR structures, however, lacking the NAD(P)H-binding motifs. The two restricted. Moreover, although the protein sequences of DDOR proteins domains of each monomer are connected by two extended loops, share the canonical FAD-binding sequence motif and conserved CXXC different to TrxR, where the domains are connected by two antiparallel motif, they lack the conserved motifs required for NAD(P)H-binding, β-strands. The relative orientation of the two domains in FFTRs is and further investigations showed that DDORs are pyridine different to the TrxR FO and FR forms. Comparison of the FFTR struc­ nucleotide-independent enzymes [132]. The same study also showed tures to the FFTR2-Trx2 crystal complex (PDBid:6GND) revealed a that although DDOR does not function as a TrxR in Trx reduction, the ◦ structural domain rearrangement in FFTR, involving a 24 rotation of redox-active CXXC motif is in direct two-electron redox communication the pseudo-NAD(P)H-binding domain relative to the FAD-binding with the flavin. Redox proteins or LMW thiols such as GSH were pro­ domain, uncovering the CXXC motif for interaction with Trx posed to function as donors of reducing equivalents to DDORs, however, (Fig. 10A). Both orientations have been described to represent the FR further experiments are needed to elucidate the true physiological donor

15 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

7. Concluding remarks

Our structural survey of published structures containing the low Mr TrxR-like fold resulted in ~30 protein types, which we classified into five groups of oxidoreductases, based on their structural similarity and degree of variation. We characterized group (1) oxidoreductases as en­ zymes containing only the low Mr TrxR-like fold, a fold serving as a conserved structural component seen in functionally unrelated oxido­ reductases. The conservation of the structural architecture among members of structural group (1) oxidoreductases illustrates how en­ zymes sharing the same structural organization can catalyze a diverse set of chemical reactions, however, modulated by small structural modifications.The rapid growth in sequence and structural information has allowed for the identification of numerous homologous TrxR-like oxidoreductases, some which have evolved peculiar features and vari­ ations to cofactor dependency. Ultimately, new biological roles and re­ action mechanisms have been assigned to these enzymes, and will likely continue to in the future. In addition to variations in the use of redox- active cofactors and motifs, and of electron donors/acceptors, the perhaps most prominent overall structural difference among these structurally related enzymes is the great variation seen in the orientation of the NAD(P)H-binding domain (or pseudo-NAD(P)H-binding domain) relative to the FAD-binding domain; a key conformational change in the Fig. 13. Different domain orientations in TrxR-like oxidoreductases. modulation of catalytic activity (summarized in a structural overlay in Structural alignment of selected TrxR-like oxidoreductases discussed in this Fig. 13). review, showing large variations in rotation of the NAD(P)H-binding domain Selected members of structural group (1) oxidoreductases have been (pseudo-NAD(P)H-binding domain in DDOR, FFTR, DTR, TRi, DepH, GliT, and discussed in detail, from a structural and functional perspective. Inter­ AclR) relative to the FAD-binding domain. Only one monomer of the homodi­ meric structures is depicted. PDBid: 1F6M (TrxR, FR), 1TDF (TrxR, FO), 6GAS estingly, several of these structurally similar oxidoreductases have (FNR2), 3LZX (FNR YumC), 3AB1 (FNR), 3ZBW (FNR), 5TWB (IruO), 7A76 evolved the capacity to function on several substrates, further illus­ (Bdr), 5N0J (DDOR), 6GND (FFTR), 5J60 (DTR), 5NII (TRi), 4JN9 (DepH), trating the extent to which evolution has experimented with fla­ 4NTC (GliT), 7BR0 (AclR). voenzymes in the evolution of redox reactions. Questions remain unanswered with respect to how specific or selective many of these of DDOR enzymes. enzymes are; if they work on a specific substrate in vivo, if they have a preferred substrate but can act on others under certain conditions, or if they are highly promiscuous and unselective. With the increasing 6.3. Thioredoxin oxidoreductase fold protein AclR knowledge on functional diversity among these TrxR- and FNR-like enzymes during the last decades, it is likely that other similar enzymes Epidithiodiketopiperazines (ETPs) comprise a structurally diverse will be discovered with alternative functions in the future. and ecologically and medically important family of mycotoxins [138]. The ETP aspirochlorine, a toxic compound produced by A. oryzae, a Acknowledgements fungus used is sake brewing, has been shown to exhibit antifungal, antibacterial, and antiviral activities [139–141], and has been suggested This study has been funded by grants from the Research Council of to have a high affinity to the main protease Mpro in the severe acute Norway (301584) and through support by the University of Oslo. respiratory syndrome coronavirus (SARS-CoV-2) [142]. The structure of aspirochlorine is unusual, due to its rearranged skeleton with a References seven-membered disulfide bridge linked to a sporoaminal ring system [143] (Fig. 12). A recent study has reported functional and structural [1] D. Leys, N.S. Scrutton, Sweating the assets of flavin cofactors: new insight of chemical versatility from knowledge of structure and mechanism, Curr. Opin. evidence of a novel type of TrxR-like oxidoreductase, AclR, catalyzing Struct. Biol. 41 (2016) 19–26. the carbon-sulfur bond migration and spiro-ring formation in the [2] R. Teufel, Flavin-catalyzed redox tailoring reactions in natural biosynthetic pathway of aspirochlorine [136]. A number of fungi, in biosynthesis, Arch. Biochem. Biophys. 632 (2017) 20–27. [3] P. Macheroux, B. Kappes, S.E. Ealick, Flavogenomics - a genomic and structural addition to A. oryzae, have been shown to carry the gene for an AclR-like view of flavin-dependent proteins, FEBS J. 278 (15) (2011) 2625–2634. enzyme in their acl-type gene clusters, suggesting that these clusters [4] M.L. Mascotti, M.J. Ayub, N. Furnham, J.M. Thornton, R.A. Laskowski, Chopping could code enzymes involved in the biosynthesis of unique compounds and changing: the evolution of the flavin-dependent monooxygenases, J. Mol. Biol. 428 (15) (2016) 3131–3146. structurally related to the rare scaffold seen in aspirochlorine. AclR is a [5] I. Sillitoe, N. Dawson, T.E. Lewis, S. Das, J.G. Lees, P. Ashford, A. Tolulope, H. homodimer composed solely of the TrxR-like fold (PDBid:7BR0) M. Scholes, I. Senatorov, A. Bujan, F.C. Rodriguez-Conde, B. Dowling, (Fig. 12A), however, lacking the NAD(P)H-binding site, and has been J. Thornton, C.A. Orengo, CATH: expanding the horizons of structure-based functional annotations for genome sequences, Nucleic Acids Res. 47 (D1) (2019) further shown to function in an NAD(P)H-independent manner using O2 D280–D284. as an electron acceptor. Moreover, bioinformatics and structural in­ [6] V. Joosten, W.J.H. van Berkel, Flavoenzymes. Curr. Opin. Chem. Biol. 11 (2) vestigations revealed that AclR contains a noncanonical CXXH motif, (2007) 195–202. distinct from the CXXC motif found in other related oxidoreductases [7] S. Placzek, I. Schomburg, A. Chang, L. Jeske, M. Ulbrich, J. Tillack, D. Schomburg, BRENDA in 2017: new perspectives and new tools in BRENDA, (Fig. 12B). Mutational analyses further confirmedthat the CXXH motif is Nucleic Acids Res. 45 (D1) (2017) D380–D388. crucial for catalysis, placing AclR in a distinct class of TrxR-like oxido­ [8] L.S. Vidal, C.L. Kelly, P.M. Mordaka, J.T. Heap, Review of NAD(P)H-dependent reductases with a specialized catalytic core. Moreover, the study sug­ oxidoreductases: properties, engineering and application, Biochim. Biophys. Acta Protein Proteonomics 1866 (2) (2018) 327–347. gests the independence of FAD to the redox reaction, however, [9] S. Ojha, E.C. Meng, P.C. Babbitt, Evolution of function in the "two dinucleotide suggesting that FAD plays a critical role as a structural stabilizing agent binding domains’" flavoproteins, PLoS Comput. Biol. 3 (7) (2007) 1268–1280. [136].

16 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

[10] N. Linzner, V.V. Loi, V.N. Fritsch, Q.N. Tung, S. Stenzel, M. Wirtz, R. Hell, C. [37] S.B. Mulrooney, C.H. Williams, Potential active-site base of thioredoxin reductase J. Hamilton, K. Tedin, M. Fulde, H. Antelmann, Staphylococcus aureus uses the from Escherichia-coli - examination of histidine(245) and aspartate(139) by site- bacilliredoxin (BrxAB)/Bacillithiol disulfide reductase (YpdA) redox pathway to directed mutagenesis, Biochemistry 33 (11) (1994) 3148–3154. defend against oxidative stress under infections, Front. Microbiol. 10 (2019) [38] R.L. Fagan, B.A. Palfey, Flavin-Dependent Enzymes, vol. 7, Elsevier, Oxford, U.K., 1355. 2010. [11] M. Rawat, J.A. Maupin-Furlow, Redox and thiols in archaea, Antioxidants 9 (5) [39] J. Kuriyan, T.S.R. Krishna, L. Wong, B. Guenther, A. Pahler, C.H. Williams, (2020), 381. P. Model, Convergent evolution of similar function in 2 structurally divergent [12] A. Argyrou, J.S. Blanchard, Flavoprotein disulfide reductases: advances in enzymes, Nature 352 (6331) (1991) 172–174. chemistry and function, in: K. Moldave (Ed.), Progress in Nucleic Acid Research [40] L. Holm, DALI and the persistence of protein shape, Protein Sci. 29 (1) (2020) and Molecular Biology, vol. 78, 2004, pp. 89–142. 128–140. [13] J. Lu, A. Holmgren, The thioredoxin antioxidant system, Free Radic. Biol. Med. 66 [41] I. Gudim, M. Hammerstad, M. Lofstad, H.-P. Hersleth, The characterization of (2014) 75–87. different flavodoxin reductase-flavodoxin (FNR-Fld) interactions reveals an [14] M. Deponte, Glutathione catalysis and the reaction mechanisms of glutathione- efficientFNR-fld redox pair and identifiesa novel FNR subclass, Biochemistry 57 dependent enzymes, Biochim. Biophys. Acta Gen. Subj. 1830 (5) (2013) (37) (2018) 5427–5436. 3217–3266. [42] Z.A. Wood, L.B. Poole, P.A. Karplus, Structure of intact AhpF reveals a mirrored [15] W.J.H. van Berkel, N.M. Kamerbeek, M.W. Fraaije, Flavoprotein thioredoxin-like active site and implies large domain rotations during catalysis, monooxygenases, a diverse class of oxidative biocatalysts, J. Biotechnol. 124 (4) Biochemistry 40 (13) (2001) 3900–3911. (2006) 670–689. [43] C.N. Jensen, S.T. Ali, M.J. Allen, G. Grogan, Exploring nicotinamide cofactor [16] C.N. Jensen, S.T. Ali, M.J. Allen, G. Grogan, Mutations of an NAD(P)H-dependent promiscuity in NAD(P)H-dependent flavin containing monooxygenases (FMOs) flavoprotein monooxygenase that influence cofactor promiscuity and using natural variation within the phosphate binding loop. Structure and activity enantioselectivity, Febs Open Bio 3 (2013) 473–478. of FMOs from Cellvibrio sp. BR and Pseudomonas stutzeri NF13, J. Mol. Catal. B [17] C.N. Jensen, J. Cartwright, J. Ward, S. Hart, J.P. Turkenburg, S.T. Ali, M.J. Allen, Enzym. 109 (2014) 191–198. G. Grogan, A flavoprotein monooxygenase that catalyses a baeyer-villiger [44] I.A. Mirza, B.J. Yachnin, S.Z. Wang, S. Grosse, H. Bergeron, A. Imura, H. Iwaki, reaction and thioether oxidation using NADH as the nicotinamide cofactor, Y. Hasegawa, P.C.K. Lau, A.M. Berghuis, Crystal structures of cyclohexanone Chembiochem 13 (6) (2012) 872–878. monooxygenase reveal complex domain movements and a sliding cofactor, J. Am. [18] P. Mulo, M. Medina, Interaction and electron transfer between ferredoxin-NADP( Chem. Soc. 131 (25) (2009) 8848–8854. +) oxidoreductase and its partners: structural, functional, and physiological [45] R. Orru, H.M. Dudek, C. Martinoli, D.E.T. Pazmino, A. Royant, M. Weik, M. implications, Photosynth. Res. 134 (3) (2017) 265–280. W. Fraaije, A. Mattevi, Snapshots of enzymatic baeyer-villiger catalysis oxygen [19] D. Seo, S. Okabe, M. Yanase, K. Kataoka, T. Sakurai, Studies of interaction of activation and intermediate stabilization, J. Biol. Chem. 286 (33) (2011) homo-dimeric ferredoxin-NAD(P)(+) oxidoreductases of Bacillus subtilis and 29284–29291. Rhodopseudomonas palustris, that are closely related to thioredoxin reductases in [46] S. Franceschini, H.L. van Beek, A. Pennetta, C. Martinoli, M.W. Fraaije, amino acid sequence, with ferredoxins and pyridine nucleotide coenzymes, A. Mattevi, Exploring the structural basis of substrate preferences in baeyer- Biochim. Biophys. Acta Protein Proteonomics 1794 (4) (2009) 594–601. villiger monooxygenases INSIGHT FROM STEROID MONOOXYGENASE, J. Biol. [20] F. Hannemann, A. Bichet, K.M. Ewen, R. Bernhardt, Cytochrome P450 systems - Chem. 287 (27) (2012) 22626–22634. biological variations of electron transport chains, Biochim. Biophys. Acta Gen. [47] J. Olucha, K.M. Meneely, A.S. Chilton, A.L. Lamb, Two structures of an N- Subj. 1770 (3) (2007) 330–344. hydroxylating flavoprotein monooxygenase ornithine hydroxylase from [21] Z.Q. Wang, R.J. Lawson, M.R. Buddha, C.C. Wei, B.R. Crane, A.W. Munro, D. Pseudomonas aeruginosa, J. Biol. Chem. 286 (36) (2011) 31789–31798. J. Stuehr, Bacterial flavodoxins support nitric oxide production by bacillus [48] T. Wagner, J. Koch, U. Ermler, S. Shima, Methanogenic heterodisulfidereductase subtilis nitric-oxide synthase, J. Biol. Chem. 282 (4) (2007) 2196–2202. (HdrABC-MvhAGD) uses two noncubane 4Fe-4S clusters for reduction, Science [22] A. Aliverti, V. Pandini, A. Pennati, M. de Rosa, G. Zanetti, Structural and 357 (6352) (2017) 699–702. functional diversity of ferredoxin-NADP(+) reductases, Arch. Biochem. Biophys. [49] M.M. Cherney, Y.F. Zhang, M. Solomonson, J.H. Weiner, M.N.G. James, Crystal 474 (2) (2008) 283–291. structure of sulfide:quinone oxidoreductase from acidithiobacillus ferrooxidans: [23] N. Muraki, D. Seo, T. Shiba, T. Sakurai, G. Kurisu, Asymmetric dimeric structure insights into sulfidotrophic respiration and detoxification, J. Mol. Biol. 398 (2) of ferredoxin-NAD(P)(+) oxidoreductase from the green sulfur bacterium (2010) 292–305. Chlorobaculum tepidum: implications for binding ferredoxin and NADP(+), [50] Q. Cheng, T. Sandalova, Y. Lindqvist, E.S.J. Arner, Crystal structure and catalysis J. Mol. Biol. 401 (3) (2010) 403–414. of the selenoprotein thioredoxin reductase 1, J. Biol. Chem. 284 (6) (2009) [24] T. Mandai, S. Fujiwara, S. Imaoka, A novel electron transport system for 3998–4008. thermostable CYP175A1 from Thermus thermophilus HB27, FEBS J. 276 (8) [51] P.A. Karplus, G.E. Schulz, Substrate binding and catalysis by glutathione- (2009) 2416–2429. reductase as derived from refined enzyme - substrate crystal-structures at 2Å [25] D. Ritz, J. Beckwith, Roles of thiol-redox pathways in bacteria, Annu. Rev. resolution, J. Mol. Biol. 210 (1) (1989) 163–180. Microbiol. 55 (2001) 21–48. [52] Y.C. Lo, T.P. Ko, W.C. Su, T.L. Su, A.H.J. Wang, Terpyridine-platinum(II) [26] A. Holmgren, B.O. Soderberg, H. Eklund, C.I. Branden, 3-dimensional structure of complexes are effective inhibitors of mammalian topoisomerases and human Escherichia-coli thioredoxin-S2 to 2.8 Å resolution, Proc. Natl. Acad. Sci. U.S.A. 72 thioredoxin reductase 1, J. Inorg. Biochem. 103 (7) (2009) 1082–1092. (6) (1975) 2305–2309. [53] R. De Gasparo, O. Halgas, D. Harangozo, M. Kaiser, E.F. Pai, R.L. Krauth-Siegel, [27] A. Holmgren, THIOREDOXIN. Annu. Rev. Biochem. 54 (1985) 237–271. F. Diederich, Targeting a large active site: structure-based design of nanomolar [28] A. Holmgren, R. Sengupta, The use of thiols by ribonucleotide reductase, Free inhibitors of trypanosoma brucei trypanothione reductase, Chem. Eur. J. 25 (49) Radic. Biol. Med. 49 (11) (2010) 1617–1628. (2019) 11416–11421. [29] C.H. Williams, L.D. Arscott, S. Muller, B.W. Lennon, M.L. Ludwig, P.F. Wang, D. [54] F. Van Petegem, D. De Vos, S. Savvides, B. Vergauwen, J. Van Beelumen, M. Veine, K. Becker, R.H. Schirmer, Thioredoxin reductase - two modes of Understanding nicotinamide dinucleotide cofactor and substrate specificity in catalysis have evolved, Eur. J. Biochem. 267 (20) (2000) 6110–6117. class I flavoprotein disulfide oxidoreductases: crystallographic analysis of a [30] T. Sandalova, L.W. Zhong, Y. Lindqvist, A. Holmgren, G. Schneider, Three- glutathione amide reductase, J. Mol. Biol. 374 (4) (2007) 883–889. dimensional structure of a mammalian thioredoxin reductase: implications for [55] R. Ledwidge, B. Patel, A.P. Dong, D. Fiedler, M. Falkowski, J. Zelikova, A. mechanism and evolution of a selenocysteine-dependent enzyme, Proc. Natl. O. Summers, E.F. Pai, S.M. Miller, NmerA, the metal binding domain of mercuric Acad. Sci. U.S.A. 98 (17) (2001) 9533–9538. ion reductase, removes Hg2+ from proteins, delivers it to the catalytic core, and [31] O. Dym, D. Eisenberg, Sequence-structure analysis of FAD-containing proteins, protects cells under glutathione-depleted conditions, Biochemistry 44 (34) (2005) Protein Sci. 10 (9) (2001) 1712–1728. 11402–11416. [32] D. Susanti, U. Loganathan, A. Compton, B. Mukhopadhyay, A reexamination of [56] G. Salinas, W. Gao, Y. Wang, M. Bonilla, L. Yu, A. Novikov, V.G. Virginio, H. thioredoxin reductase from thermoplasma acidophilum, a thermoacidophilic B. Ferreira, M. Vieites, V.N. Gladyshev, D. Gambino, S.D. Dai, The enzymatic and euryarchaeon, identifies it as an NADH-dependent enzyme, ACS Omega 2 (8) structural basis for inhibition of echinococcus granulosus thioredoxin glutathione (2017) 4180–4187. reductase by gold(I), Antioxidants Redox Signal. 27 (18) (2017) 1491–1504. [33] B.W. Lennon, C.H. Williams, M.L. Ludwig, Twists in catalysis: alternating [57] G. Hagelueken, L. Wiehlmann, T.M. Adams, H. Kolmar, D.W. Heinz, B. Tummler, conformations of Escherichia coli thioredoxin reductase, Science 289 (5482) W.D. Schubert, Crystal structure of the electron transfer complex rubredoxin- (2000) 1190–1194. rubredoxin reductase of Pseudomonas aeruginosa, Proc. Natl. Acad. Sci. U.S.A. [34] B.W. Lennon, C.H. Williams, M.L. Ludwig, Crystal structure of reduced 104 (30) (2007) 12276–12281. thioredoxin reductase from Escherichia coli: structural flexibility in the [58] I.F. Sevrioukova, Structure/function relations in AIFM1 variants associated with isoalloxazine ring of the flavin adenine dinucleotide cofactor, Protein Sci. 8 (11) neurodegenerative disorders, J. Mol. Biol. 428 (18) (2016) 3650–3665. (1999) 2366–2379. [59] I.F. Sevrioukova, H.Y. Li, T.L. Poulos, Crystal structure of putidaredoxin reductase [35] C.H. Williams, Flavoprotein structure and mechanism. 6. Mechanism and from Pseudomonas putida, the final structural component of the cytochrome structure of thioredoxin reductase from Escherichia-coli, Faseb. J. 9 (13) (1995) P450cam monooxygenase, J. Mol. Biol. 336 (4) (2004) 889–902. 1267–1276. [60] R. Friemann, K. Lee, E.N. Brown, D.T. Gibson, H. Eklund, S. Ramaswamy, [36] G. Waksman, T.S.R. Krishna, C.H. Williams, J. Kuriyan, Crystal structure of Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase Escherichia-coli thioredoxin reductase refined at 2-Ångstrom¨ resolution - enzyme system, Acta Crystallogr. Sect. D Biol. Crystallogr. 65 (2009) 24–33. implications for a large conformational change during catalysis, J. Mol. Biol. 236 [61] M. Senda, S. Kishigami, S. Kimura, M. Fukuda, T. Ishida, T. Senda, Molecular (3) (1994) 800–816. mechanism of the redox-dependent interaction between NADH-dependent ferredoxin reductase and rieske-type 2Fe-2S ferredoxin, J. Mol. Biol. 373 (2) (2007) 382–400.

17 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

[62] E.J. Crane, J.I. Yeh, J. Luba, A. Claiborne, Analysis of the kinetic and redox [89] S.A. Loutet, M.J. Kobylarz, C.H.T. Chau, M.E.P. Murphy, IruO is a reductase for properties of the NADH peroxidase R303M mutant: correlation with the crystal heme degradation by IsdI and IsdG proteins in Staphylococcus aureus, J. Biol. structure, Biochemistry 39 (34) (2000) 10353–10364. Chem. 288 (36) (2013) 25749–25759. [63] D.V. Titov, V. Cracan, R.P. Goodman, J. Peng, Z. Grabarek, V.K. Mootha, [90] M. Hammerstad, I. Gudim, H.-P. Hersleth, The crystal structures of bacillithiol Complementation of mitochondrial electron transport chain by manipulation of disulfide reductase Bdr (YpdA) provide structural and functional insight into a the NAD(+)/NADH ratio, Science 352 (6282) (2016) 231–235. new type of FAD-containing NADPH-dependent oxidoreductase, Biochemistry 59 [64] T. Stehle, A. Claiborne, G.E. Schulz, NADH binding-site and catalysis of NADH (51) (2020) 4793–4798. peroxidase, Eur. J. Biochem. 211 (1–2) (1993) 221–226. [91] I.V. Mikheyeva, J.M. Thomas, S.L. Kolar, A.R. Corvaglia, N. Gaiotaa, S. Leo, [65] B.D. Wallace, J.S. Edwards, J.R. Wallen, W.J.A. Moolman, R. van der P. Francois, G.Y. Liu, M. Rawat, A.L. Cheung, YpdA, a putative bacillithiol Westhuyzen, E. Strauss, M.R. Redinbo, A. Claiborne, Turnover-dependent disulfide reductase, contributes to cellular redox homeostasis and virulence in covalent inactivation of Staphylococcus aureus coenzyme A-disulfide reductase Staphylococcus aureus, Mol. Microbiol. 11 (2019) 1039–1056. by coenzyme A-mimetics: mechanistic and structural insights, Biochemistry 51 [92] V.G. Fowler, J.M. Miro, B. Hoen, C.H. Cabell, E. Abrutyn, E. Rubinstein, G. (39) (2012) 7699–7711. R. Corey, D. Spelman, S.F. Bradley, B. Barsic, P.A. Pappas, K.J. Anstrom, D. Wray, [66] D. Seo, H. Sakurai, Purification and characterization of ferredoxin-NAD(P)(+) C.Q. Fortes, I. Anguera, E. Athan, P. Jones, J.T.M. van der Meer, T.S.J. Elliott, D. reductase from the green sulfur bacterium Chlorobium tepidum, Biochim. P. Levine, A.S. Bayer, Staphylococcus aureus endocarditis - a consequence of Biophys. Acta Protein Struct. Mol. Enzymol. 1597 (1) (2002) 123–132. medical progress, JAMA, J. Am. Med. Assoc. 293 (24) (2005) 3012–3021. [67] D. Seo, K. Kamino, K. Inoue, H. Sakurai, Purification and characterization of [93] R.M. Klevens, M.A. Morrison, J. Nadle, S. Petit, K. Gershman, S. Ray, L. ferredoxin-NADP(+) reductase encoded by Bacillus subtilis yumC, Arch. H. Harrison, R. Lynfield, G. Dumyati, J.M. Townes, A.S. Craig, E.R. Zell, G. Microbiol. 182 (1) (2004) 80–89. E. Fosheim, L.K. McDougal, R.B. Carey, S.K. Fridkin, A.B.M. Investigators, [68] M. Lofstad, I. Gudim, M. Hammerstad, Å.K. Røhr, H.-P. Hersleth, Activation of the Invasive methicillin-resistant Staphylococcus aureus infections in the United class Ib ribonucleotide reductase by a flavodoxin reductase in Bacillus cereus, States, JAMA, J. Am. Med. Assoc. 298 (15) (2007) 1763–1771. Biochemistry 55 (36) (2016) 4998–5001. [94] J.E. Cassat, E.P. Skaar, Iron in infection and immunity, Cell Host Microbe 13 (5) [69] Z. Yan, Y.W. Nam, S. Fushinobu, T. Wakagi, Sulfolobus tokodaii ST2133 is (2013) 510–520. characterized as a thioredoxin reductase-like ferredoxin:NADP(+) [95] J.J. Bullen, H.J. Rogers, P.B. Spalding, C.G. Ward, Iron and infection: the heart of oxidoreductase, Extremophiles 18 (1) (2014) 99–110. the matter, FEMS Immunol. Med. Microbiol. 43 (3) (2005) 325–330. [70] H. Komori, D. Seo, T. Sakurai, Y. Higuchi, Crystal structure analysis of Bacillus [96] J.C. Grigg, G. Ukpabi, C.F.M. Gaudin, M.E.P. Murphy, Structural biology of heme subtilis ferredoxin-NADP(+) oxidoreductase and the structural basis for its binding in the Staphylococcus aureus Isd system, J. Inorg. Biochem. 104 (3) substrate selectivity, Protein Sci. 19 (12) (2010) 2279–2290. (2010) 341–348. [71] S.B. Mulrooney, C.H. Williams, Evidence for two conformational states of [97] N. Muryoi, M.T. Tiedemann, M. Pluym, J. Cheung, D.E. Heinrichs, M.J. Stillman, thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic Demonstration of the iron-regulated surface determinant (Isd) heme transfer quenchers of flavin fluorescence as probes to observe domain rotation, Protein pathway in Staphylococcus aureus, J. Biol. Chem. 283 (42) (2008) 28125–28136. Sci. 6 (10) (1997) 2188–2195. [98] S.K. Mazmanian, E.P. Skaar, A.H. Gaspar, M. Humayun, P. Gornicki, J. Jelenska, [72] C.C. Moser, J.L.R. Anderson, P.L. Dutton, Guidelines for tunneling in enzymes, A. Joachmiak, D.M. Missiakas, O. Schneewind, Passage of heme-iron across the Biochim. Biophys. Acta Bioenerg. 1797 (8) (2010) 1573–1586. envelope of Staphylococcus aureus, Science 299 (5608) (2003) 906–909. [73] M.J. Kobylarz, G.A. Heieis, S.A. Loutet, M.E.P. Murphy, Iron uptake [99] V.J. Torres, G. Pishchany, M. Humayun, O. Schneewind, E.P. Skaar, oxidoreductase (IruO) uses a flavin adenine dinucleotide semiquinone Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron intermediate for iron-siderophore reduction, ACS Chem. Biol. 12 (7) (2017) utilization, J. Bacteriol. 188 (24) (2006) 8421–8429. 1778–1786. [100] C.L. Vermeiren, M. Pluym, J. Mack, D.E. Heinrichs, M.J. Stillman, [74] P. Reichard, Enzymatic synthesis of deoxyribonucleotides. 1. formation of Characterization of the heme binding properties of Staphylococcus aureus IsdA, deoxycytidine diphosphate from cytidine diphosphate with enzymes from Biochemistry 45 (42) (2006) 12867–12875. Escherichia coli, J. Biol. Chem. 237 (11) (1962) 3513–3519. [101] G. Pishchany, S.E. Dickey, E.P. Skaar, Subcellular localization of the [75] N. Nordlund, P. Reichard, Ribonucleotide reductases, Annu. Rev. Biochem. 75 Staphylococcus aureus heme iron transport components IsdA and IsdB, Infect. (2006) 681–706. Immun. 77 (7) (2009) 2624–2634. [76] K.K. Andersson (Ed.), Ribonucleotide Reductase, Nova Science Publishers, Inc., [102] M.T. Tiedemann, D.E. Heinrichs, M.J. Stillman, Multiprotein heme shuttle Hauppauge, NY, USA, 2008. pathway in Staphylococcus aureus: iron-regulated surface determinant cog-wheel [77] J.A. Cotruvo, J. Stubbe, An active dimanganese(III)-Tyrosyl radical cofactor in kinetics, J. Am. Chem. Soc. 134 (40) (2012) 16578–16585. Escherichia coli class Ib ribonucleotide reductase, Biochemistry 49 (6) (2010) [103] A. Dryla, D. Gelbmann, A. von Gabain, E. Nagy, Identification of a novel iron 1297–1309. regulated staphylococcal surface protein with haptoglobin-haemoglobin binding [78] J.A. Cotruvo, J. Stubbe, Escherichia coli class Ib ribonucleotide reductase contains activity, Mol. Microbiol. 49 (1) (2003) 37–53. a dimanganese(III)-Tyrosyl radical cofactor in vivo, Biochemistry 50 (10) (2011) [104] J.C. Grigg, C.L. Vermeiren, D.E. Heinrichs, M.E.P. Murphy, Heme coordination by 1672–1681. Staphylococcus aureus IsdE, J. Biol. Chem. 282 (39) (2007) 28815–28822. [79] A.K. Boal, J.A. Cotruvo, J. Stubbe, A.C. Rosenzweig, Structural basis for [105] M. Pluym, C.L. Vermeiren, J. Mack, D.E. Heinrichs, M.J. Stillman, Heme binding activation of class Ib ribonucleotide reductase, Science 329 (5998) (2010) properties of staphylococcus aureus lsdE, Biochemistry 46 (44) (2007) 1526–1530. 12777–12787. [80] J. Chen, J. Shen, C. Solem, P.R. Jensen, A new type of YumC-like ferredoxin [106] E.P. Skaar, A.H. Gaspar, O. Schneewind, IsdG and IsdI, heme-degrading enzymes (flavodoxin)reductase is involved in ribonucleotide reduction, mBio 6 (6) (2015), in the cytoplasm of Staphylococcus aureus, J. Biol. Chem. 279 (1) (2004) e01132-15. 436–443. [81] L. McIver, C. Leadbeater, D.J. Campopiano, R.L. Baxter, S.N. Daff, S.K. Chapman, [107] R.Y. Wu, E.P. Skaar, R.G. Zhang, G. Joachimiak, P. Gornicki, O. Schneewind, A.W. Munro, Characterisation of flavodoxin NADP(+) oxidoreductase and A. Joachimiak, Staphylococcus aureus IsdG and IsdI, heme-degrading enzymes flavodoxin; key components of electron transfer in Escherichia coli, Eur. J. with structural similarity to monooxygenases, J. Biol. Chem. 280 (4) (2005) Biochem. 257 (3) (1998) 577–585. 2840–2846. [82] D. Seo, T. Asano, H. Komori, T. Sakurai, Role of the C-terminal extension stacked [108] F.C. Beasley, D.E. Heinrichs, Siderophore-mediated iron acquisition in the on the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide staphylococci, J. Inorg. Biochem. 104 (3) (2010) 282–288. prosthetic group in ferredoxin-NADP(+) oxidoreductase from Bacillus subtilis, [109] F.C. Beasley, C.L. Marolda, J. Cheung, S. Buac, D.E. Heinrichs, Staphylococcus Plant Physiol. Biochem. 81 (2014) 143–148. aureus transporters hts, sir, and sst capture iron liberated from human transferrin [83] D. Seo, T. Asano, C-terminal residues of ferredoxin-NAD(P)(+) reductase from by staphyloferrin A, staphyloferrin B, and catecholamine stress hormones, Chlorobaculum tepidum are responsible for reaction dynamics in the hydride respectively, and contribute to virulence, Infect. Immun. 79 (6) (2011) transfer and redox equilibria with NADP(+)/NADPH, Photosynth. Res. 136 (3) 2345–2355. (2018) 275–290. [110] M.T. Sebulsky, D. Hohnstein, M.D. Hunter, D.E. Heinrichs, Identification and [84] F. Meschi, F. Wiertz, L. Klauss, A. Blok, B. Ludwig, A. Merli, H.A. Heering, G. characterization of a membrane permease involved in iron-hydroxamate L. Rossi, M. Ubbink, Efficient electron transfer in a protein network lacking transport in Staphylococcus aureus, J. Bacteriol. 182 (16) (2000) 4394–4400. specific interactions, J. Am. Chem. Soc. 133 (42) (2011) 16861–16867. [111] M.T. Sebulsky, D.E. Heinrichs, Identification and characterization of fhuD1 and [85] D. Seo, A. Tomioka, N. Kusumoto, M. Kamo, I. Enami, H. Sakurai, Purification of fhuD2, two genes involved in iron-hydroxamate uptake in Staphylococcus aureus, ferredoxins and their reaction with purified reaction center complex from the J. Bacteriol. 183 (17) (2001) 4994–5000. green sulfur bacterium Chlorobium tepidum, Biochim. Biophys. Acta Bioenerg. [112] R.C. Hider, X.L. Kong, Chemistry and biology of siderophores, Nat. Prod. Rep. 27 1503 (3) (2001) 377–384. (5) (2010) 637–657. [86] J.K. Holden, N. Lim, T.L. Poulos, Identification of redox partners and [113] M. Hannauer, A.J. Arifin, D.E. Heinrichs, Involvement of reductases IruO and development of a novel chimeric bacterial nitric oxide synthase for structure NtrA in iron acquisition by Staphylococcus aureus, Mol. Microbiol. 96 (6) (2015) activity analyses, J. Biol. Chem. 289 (42) (2014) 29437–29445. 1192–1210. [87] N.U. Frigaard, A.G.M. Chew, H. Li, J.A. Maresca, D.A. Bryant, Chlorobium [114] N. Archipowa, R.J. Kutta, D.J. Heyes, N.S. Scrutton, Stepwise hydride transfer in a tepidum: insights into the structure, physiology, and metabolism of a green sulfur biological system: insights into the reaction mechanism of the light-dependent bacterium derived from the complete genome sequence, Photosynth. Res. 78 (2) protochlorophyllide oxidoreductase, Angew. Chem. Int. Ed. 57 (10) (2018) (2003) 93–117. 2682–2686. [88] E.P. Skaar, O. Schneewind, Iron-regulated surface determinants (Isd) of [115] M. Bose, S. Bhattacharyya, R. Biswas, A. Roychowdhury, A. Bhattacharjee, A. Staphylococcus aureus: stealing iron from heme, Microb. Infect. 6 (4) (2004) K. Ghosh, A.K. Das, Elucidation of the mechanism of disulfide exchange between 390–397.

18 M. Hammerstad and H.-P. Hersleth Archives of Biochemistry and Biophysics 702 (2021) 108826

staphylococcal thioredoxin2 and thioredoxin reductase2: a structural insight, pyridine nucleotide-independent thioredoxin reductases, Proc. Natl. Acad. Sci. U. Biochimie 160 (2019) 1–13. S.A. 115 (51) (2018) 12967–12972. [116] N. Couto, J. Wood, J. Barber, The role of glutathione reductase and related [132] R.M. Buey, J.B. Arellano, L. Lopez-Maury, S. Galindo-Trigo, A. Velazquez- enzymes on cellular redox homoeostasis network, Free Radic. Biol. Med. 95 Campoy, J.L. Revuelta, J.M. de Pereda, F.J. Florencio, P. Schurmann, B. (2016) 27–42. B. Buchanan, M. Balsera, Unprecedented pathway of reducing equivalents in a [117] K. Van Laer, C.J. Hamilton, J. Messens, Low-molecular-weight thiols in thiol- diflavin-linked disulfide oxidoreductase, Proc. Natl. Acad. Sci. U.S.A. 114 (48) disulfide exchange, Antioxidants Redox Signal. 18 (13) (2013) 1642–1653. (2017) 12725–12730. [118] P. Chandrangsu, V.V. Loi, H. Antelmann, J.D. Helmann, The role of bacillithiol in [133] B. Li, C.T. Walsh, Streptomyces clavuligerus HlmI is an intramolecular disulfide- gram-positive Firmicutes, Antioxidants Redox Signal. 28 (6) (2018) 445–462. forming dithiol oxidase in holomycin biosynthesis, Biochemistry 50 (21) (2011) [119] P. Ghezzi, Protein glutathionylation in health and disease, Biochim. Biophys. Acta 4615–4622. Gen. Subj. 1830 (5) (2013) 3165–3172. [134] D.H. Scharf, N. Remme, T. Heinekamp, P. Hortschansky, A.A. Brakhage, [120] V.V. Loi, M. Rossius, H. Antelmann, Redox regulation by reversible protein S- C. Hertweck, Transannular disulfide formation in gliotoxin biosynthesis and its thiolation in bacteria, Front. Microbiol. 6 (2015) 187. role in self-resistance of the human pathogen Aspergillus fumigatus, J. Am. Chem. [121] M. Imber, A.J. Pietrzyk-Brzezinska, H. Antelmann, Redox regulation by reversible Soc. 132 (29) (2010) 10136–10141. protein S-thiolation in Gram-positive bacteria, Redox Biol 20 (2019) 130–145. [135] S.D. Dai, R. Friemann, D.A. Glauser, F. Bourquin, W. Manieri, P. Schurmann, [122] J.W. Lee, S. Soonsanga, J.D. Helmann, A complex thiolate switch regulates the H. Eklund, Structural snapshots along the reaction pathway of ferredoxin- Bacillus subtilis organic peroxide sensor, OhrR. Proc. Natl. Acad. Sci. U. S. A. 104 thioredoxin reductase, Nature 448 (7149) (2007) 92–102. (21) (2007) 8743–8748. [136] Y. Tsunematsu, N. Maeda, M. Sato, K. Hara, H. Hashimoto, K. Watanabe, [123] B.K. Chi, K. Gronau, U. Mader, B. Hessling, D. Becher, H. Antelmann, S- C. Hertweck, Specialized flavoproteinpromotes sulfur migration and spiroaminal bacillithiolation protects against hypochlorite stress in Bacillus subtilis as formation in aspirochlorine biosynthesis, J. Am. Chem. Soc. 143 (1) (2021) revealed by transcriptomics and redox proteomics, Mol. Cell. Proteomics 10 (11) 206–213. (2011). M111.009506. [137] K.E. Hammel, K.L. Cornwell, B.B. Buchanan, Ferredoxin flavoprotein-linked [124] B.K. Chi, A.A. Roberts, T.T.T. Huyen, K. Basell, D. Becher, D. Albrecht, C. pathway for the reduction of thioredoxin, Proc. Natl. Acad. Sci. U.S.A. 80 (12) J. Hamilton, H. Antelmann, S-bacillithiolation protects conserved and essential (1983) 3681–3685. proteins against hypochlorite stress in Firmicutes bacteria, Antioxidants Redox [138] D.M. Gardiner, P. Waring, B.J. Howlett, The epipolythiodioxopiperazine (ETP) Signal. 18 (11) (2013) 1273–1295. class of fungal toxins: distribution, mode of action, functions and biosynthesis, [125] M. Imber, V.V. Loi, S. Reznikov, V.N. Fritsch, A.J. Pietrzyk-Brzezinska, J. Prehn, Microbiology 151 (2005) 1021–1032. C. Hamilton, M.C. Wahl, A.K. Bronowska, H. Antelmann, The aldehyde [139] D.H. Berg, R.P. Massing, M.M. Hoehn, L.D. Boeck, R.L. Hamill, A30641, a new dehydrogenase AldA contributes to the hypochlorite defense and is redox- epidithiodiketopiperazine with antifungal activity, J. Antibiot. 29 (4) (1976) controlled by protein S-bacillithiolation in Staphylococcus aureus, Redox Biol 15 394–397. (2018) 557–568. [140] P. Klausmeyer, T.G. McCloud, K.D. Tucker, J.H. Cardellina, R.H. Shoemaker, [126] B.W. Lennon, C.H. Williams, Reductive half-reaction of thioredoxin reductase Aspirochlorine class compounds from Aspergillus flavus inhibit azole-resistant from Escherichia coli, Biochemistry 36 (31) (1997) 9464–9477. Candida albicans, J. Nat. Prod. 68 (8) (2005) 1300–1302. [127] D.H. Scharf, M. Groll, A. Habel, T. Heinekamp, C. Hertweck, A.A. Brakhage, E. [141] F. Monti, F. Ripamonti, S.P. Hawser, K. Islam, Aspirochlorine, A highly selective M. Huber, Flavoenzyme-catalyzed formation of disulfide bonds in natural and potent inhibitor of fungal protein synthesis, J. Antibiot. 52 (3) (1999) products, Angew. Chem. Int. Ed. 53 (8) (2014) 2221–2224. 311–318. [128] J. Li, C. Wang, Z.M. Zhang, Y.Q. Cheng, J.H. Zhou, The structural basis of an [142] P. Singh, A. Sharma, S.P. Nandi, Identification of potent inhibitors of covid-19 NADP(+)-independent dithiol oxidase in FK228 biosynthesis, Sci. Rep. 4 (2014), main protease emzyme by molecular docking study, ChemRxiv, Preprint at, 4145. https://doi.org/10.26434/chemrxiv.12179202.v1, 2020. [129] R.M. Buey, S. Galindo-Trigo, L. Lopez-Maury, A. Velazquez-Campoy, J. [143] M.L. Zhu, X.W. Zhang, X.N. Huang, H.T. Wang, K. Anjum, Q.Q. Gu, T.J. Zhu, G. L. Revuelta, F.J. Florencio, J.M. de Pereda, P. Schurmann, B.B. Buchanan, J. Zhang, D.H. Li, Irregularly bridged epipolythiodioxopiperazines and related M. Balsera, A new member of the thioredoxin reductase family from early analogues: sources, structures, and biological activities, J. Nat. Prod. 83 (6) oxygenic photosynthetic organisms, Mol. Plant 10 (1) (2017) 212–215. (2020) 2045–2053. [130] O. Valette, T.T.T. Tran, C. Cavazza, E. Caudeville, G. Brasseur, A. Dolla, E. Talla, [144] D. Kozakov, D.R. Hall, B. Xia, K.A. Porter, D. Padhorny, C. Yueh, D. Beglov, L. Pieulle, Biochemical function, molecular structure and evolution of an atypical S. Vajda, The ClusPro web server for protein-protein docking, Nat. Protoc. 12 (2) thioredoxin reductase from Desulfovibrio vulgaris, Front. Microbiol. 8 (2017), (2017) 255–278. 1855. [145] A. Waterhouse, M. Bertoni, S. Bienert, G. Studer, G. Tauriello, R. Gumienny, F. [131] R.M. Buey, D. Fernandez-Justel, J.M. de Pereda, J.L. Revuelta, P. Schurmann, B. T. Heer, T.A.P. de Beer, C. Rempfer, L. Bordoli, R. Lepore, T. Schwede, SWISS- B. Buchanan, M. Balsera, Ferredoxin-linked flavoenzyme defines a family of MODEL: homology modelling of protein structures and complexes, Nucleic Acids Res. 46 (W1) (2018) W296–W303.

19