Crystal structure of the Siderophore-interacting protein SIP from Aeromonas hydrophila

Biochemical and Biophysical Research Communications 519 (2019) 23e28

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Crystal structure of the Siderophore-interacting protein SIP from Aeromonas hydrophila Fei Shang a, b, 2, Jing Lan a, b, 2, Lulu Wang a, b, c, Wei Liu a, b, Yuanyuan Chen a, b, Jinli Chen a, b, 1, Nam-Chul Ha d, Chunshan Quan a, b, ***, Ki Hyun Nam e, f, **, Yongbin Xu a, b, * a

Department of Bioengineering, College of Life Science, Dalian Minzu University, Dalian, 116600, Liaoning, China Key Laboratory of Biotechnology and Bioresources Utilization (Dalian Minzu University), Ministry of Education, China School of Life Science and Biotechnology, Dalian University of Technology, No 2 Linggong Road, Dalian, 116024, Liaoning, China d Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Gwanak-gu, Seoul 08826, Republic of Korea e Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea f Institute of Life Science and Natural Resources, Korea University, Seoul, 02841, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 August 2019 Accepted 14 August 2019 Available online 30 August 2019

Siderophores acquire iron from hosts under iron-limiting conditions and play an essential role in the survival of microorganisms. Siderophore-interacting proteins (SIPs) from microbes release iron from the siderophore complex by reducing ferric iron to ferrous iron, but the molecular mechanism of iron reduction remains unclear. To better understand the molecular mechanism of SIPs, we herein report the crystal structure of Aeromonas hydrophila SIP (AhSIP) in complex with flavin adenine dinucleotide (FAD) as a cofactor. AhSIP consists of an N-terminal FAD binding domain and a C-terminal NADH binding domain, which are connected by a linker region. AhSIP showed unique structural differences in the orientation of the cofactor binding lobes when compared with SIP homologs. This study identified a cluster of three basic residues (Lys48, His259 and Arg262) in AhSIP distributed around a potential substrate binding pocket. In addition, AhSIP, containing the NADH binding motif E(L)VL-X3-GE, belongs to the group I subfamily. Our results show the diverse cofactor and substrate binding sites of the SIP family. © 2019 Elsevier Inc. All rights reserved.

Keywords: Aeromonas hydrophila Siderophore-interacting proteins (SIPs) Ferric siderophore reductase (FSR)

1. Introduction Iron is a necessary element for bacterial survival and is utilized to catalyze a wide variety of indispensable enzymatic reactions [1,2]. As a transition metal, iron participates in the transfer of electrons via oxidation-reduction reactions, which results in the

* Corresponding author. Department of Bioengineering, College of Life Science, Dalian Minzu University, Dalian, 116600, Liaoning, China. ** Corresponding author. Institute of Life Science and Natural Resources, Korea University, Seoul, 02841, Republic of Korea. *** Corresponding author. Department of Bioengineering, College of Life Science, Dalian Minzu University, Dalian, 116600, Liaoning, China. E-mail addresses: [email protected] (C. Quan), [email protected] (K.H. Nam), [email protected] (Y. Xu). 1 State Key Laboratory of Fine Chemical Engineering and School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116600, Liaoning, China. 2 Both authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.08.085 0006-291X/© 2019 Elsevier Inc. All rights reserved.

existence of iron in two oxidation states, ferrous (2þ) and ferric (3þ), for the donation and acceptance of electrons, respectively [3,4]. In response to a low [Fe3þ/2þ] microenvironment, microorganisms have developed iron chelators known as siderophores, which are small molecular compounds that form tight and stable complexes with ferric iron that are used to transport the ferric siderophore across cell membranes [5,6]. Since their discovery, over 500 examples of different siderophores have been discovered [5]. When the ferric siderophore complex is absorbed by cells, only the free iron released from the complex can be further used [7]. In the process of releasing iron, it is necessary to hydrolyze the siderophore by means of esterases or reduce the ferric iron to ferrous iron, which is much less strongly chelated by the siderophore [7]. Siderophore-interacting proteins (SIPs) play a vital role in the iron acquisition of many microorganisms, such as fungi and bacteria [8]. In bacteria, the SIP superfamily is composed of two different families, the ferric siderophore reductase (FSR) family and the SIP

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F. Shang et al. / Biochemical and Biophysical Research Communications 519 (2019) 23e28

family, which are widely present several bacterial genera [9,10]. SIPs can be divided into two categories: one category generates reduced free flavins in the cytosol that then reduce the ferric siderophores, and the other type of SIP primarily utilizes NADH and/or NADPH as a reducing agent and contains a stable attachment of flavin [10]. In addition, the SIP family can be further divided into two subgroups based on differences between the C-terminal and N-terminal amino acid sequences [11]. SIPs of subgroup I have prominent C-terminal alpha-helical elements and utilize NADH as an electron donor [12,13]. Subgroup II has N-terminal sequence extensions and uses NADPH as the electron donor [12,13]. To further investigate the structure-function relationship of SIPs, we determined the crystal structure of the SIP from Aeromonas hydrophila (AhSIP) at 2.5 Å resolution. AhSIP contains the prominent C-terminal alpha-helix and belongs to subgroup I; the protein that utilizes NADH. Crucial structural differences were observed in the region of the active site and the FAD binding site, presumably rendering AhSIP inactive as a siderophore reductase. Our results provide insight into the understanding of the molecular mechanism of the SIP superfamily. 2. Materials and methods 2.1. Protein expression and purification The target sequences of AhSIP (UniProt: A0A2R4TY37) were amplified from the genomic DNA of A. hydrophila by polymerase chain reaction (PCR). The PCR products were digested by the EcoRI and HindIII restriction enzymes and subcloned into the pPROEXHTA vector (Invitrogen, USA), which contained a hexa-histidine tag at the N-terminus and a tobacco etch virus (TEV) protease recognition site between the expression tags and AhSIP. The resulting expression vector pPROEX-HTA-AhSIP was transformed into an Escherichia coli BL21 (DE3) strain. The cells were grown in LB medium containing 100 mg ml1 ampicillin at 37  C for an additional 12 h and then washed three times with M9 medium. Then, the cells were further cultured at 37  C in M9 medium containing glucose, other essential amino acids and VB1 (thiamine) until an OD600 of 0.6e0.8 was achieved. Twenty minutes later, 1 mM isopropyl-b-D-thiogalactoside (IPTG) and L-(þ)-selenomethionine were added, and the culture was further incubated for 9 h at 30  C before harvesting. Cells were harvested by centrifugation and then resuspended in buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM b-mercaptoethanol followed by disruption by sonication. The cell debris was removed by centrifugation at 13,000 rpm for 30 min at 4  C, and the lysate was loaded onto NiNTA affinity resin (GE Healthcare, USA). After washing with buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 30 mM imidazole), the target proteins were eluted with the same buffer containing 250 mM imidazole, and the hexa-histidine tag was removed by the recombinant TEV protease at 23  C for 13 h. The resulting protein of interest was diluted 3-fold with buffer (20 mM Tris-HCl, pH 8.0 and 2 mM b-mercaptoethanol) and then purified using the HiTrap Q column (GE Healthcare, USA) in a linear gradient from 0 M to 1 M NaCl with 20 mM Tris-HCl (pH 8.0) and 2 mM b-mercaptoethanol. Finally, the target protein was collected and further purified using a HiLoad Superdex 200 26/60 column (GE Healthcare) with buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 2 mM bmercaptoethanol. The purified protein was pooled and concentrated to 30 mg ml1 using a Vivaspin centrifugal concentrator (Millipore, USA) for further experiments. 2.2. Size-exclusion chromatography To determine the oligomeric state of AhSIP, 500 mL of AhSIP

(1 mg ml1) was injected into a Superdex 200 10/600 GL column (GE Healthcare, USA) equilibrated with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 2 mM b-mercaptoethanol at a flow rate of 0.5 ml min1. The molecular weights of the eluted samples were calculated based on the calibration curves by standard samples, such as cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), albumin (66 kDa), and alcohol dehydrogenase (150 kDa). 2.3. Crystallization and data collection Crystallization of the purified AhSIP was performed with the Crystal Screen HT high-throughput sparse-matrix screening kit (Hampton Research, USA) using the sitting-drop vapor diffusion method at 14  C. The SeMet-substituted crystals of AhSIP appeared with the initial conditions of mixing a 0.5 mL protein sample with an equal volume of reservoir solution (0.2 M lithium sulfate, 30% (w/v) polyethylene glycol 4000 and 0.1 M Tris-HCl, pH 8.5). The crystal was transferred to a cryoprotectant solution containing reservoir solution plus 25% (v/v) glycerol for 5 s before being flash-cooled in liquid nitrogen. The X-ray diffraction data were collected at 173  C using beamline 5C with an ADSC Q315 CCD detector at the Pohang Light Source (PLS, Pohang, Republic of Korea). The diffraction datasets were integrated and scaled using the HKL2000 software suite [14]. 2.4. Structure solution and refinement The single-wavelength anomalous dispersion (SAD) phasing method was used to obtain the initial phase of SeMet-substituted AhSIP. The SAD phasing method was used to obtain the initial phase of SeMet-substituted AhSIP using the AutoSol Program. Then, the initial model was built using PHENIX AutoBuild [14,15]. Model building was performed with the WinCoot program [16]. The final model was refined by Phenix [17]. The refinement statistics are given in Table 1. All figures were generated using the program PyMOL [18]. Homolog proteins were searched for with the DaliLite server [19]. The PISA server was used to calculate the interface areas of the oligomer [20]. Multiple sequence alignments were generated by the Clustal Omega server [21] and drawn by ESPript 3.0 [22]. The atomic coordinates and structural factors for AhSIP have been deposited in the RCSB Protein Data Bank under accession code 6K2L. 3. Results and discussion 3.1. Overall structure of AhSIP To better understand the molecular mechanism of cytosolic SIPs, we solved the crystal structure of the SIP from A. hydrophila at 2.5 Å resolution with Rwork and Rfree values of 0.20 and 0.26, respectively. The AhSIP molecule is composed of two binding domains (Fig. 1A): an N-terminal FAD binding domain (Met1-Leu122) and a C-terminal NADH binding domain (Tyr139-Ser268) that are connected by a linker peptide (Ile123-His138) between the b7-and b8-strands (Fig. 1A). A deep cleft is formed between the FAD and NADH binding domains (Fig. 1). The FAD binding domain contains a sevenstranded antiparallel b-barrel architecture (Fig. 1). The NADH binding domain (residues His138-Ser268) was composed of an a/bsandwich structure with a central five-stranded b-sheet surrounded by four a-helices (Fig. 1). There are two AhSIP molecules in the asymmetric unit, which are regarded as two monomeric molecules (named AhSIP-A and AhSIP-B, respectively) that do not significantly interact with each other (Fig. 1B). These crystallographic results are consistent with the analysis of the size exclusion chromatography results, showing that AhSIP exists in a monomeric

F. Shang et al. / Biochemical and Biophysical Research Communications 519 (2019) 23e28 Table 1 Crystallography data and refinement statistics. Data sets Beamline Resolution range (Å) Space group Total/unique reflections a, b, c (Å) a, b, g (o) Rsym (%) Completeness (%) Multiplicity Average I/s(I) Model refinement bRfactor/Rfree (%) No. of protein atoms No. of water molecule Average B factor (Å2) R.m.s.d (Bond) R.m.s.d (Angles) Ramachandran Preferred (%) Ramachandran Outliers (%) PDB code

Beamline 5C at PLS 35.83e2.5 (2.589e2.5)a C2221 19143/1771 130.402, 149.991, 55.896 90.00, 90.00, 90.00 12.5 (55.6) 98.51 (92.58) 9.2 (5.1) 12.96 (2.27) 20.41/26.09 3465 154 33.0 0.009 1.09 99.78 0.22 6K2L

a

Values in the parentheses refers to the highest resolution shell. P hkljFo-Fcj/ hkljFoj for all data with Fo > 2s(Fo), excluding data used to calculate P P Rfree. xRfree ¼ hkljFo-Fcj/ hkljFoj for all data with Fo > 2s (Fo) that were excluded from refinement

bP

state in solution (Supporting Fig. S1). In AhSIP-A, the a2-b6 loop and a8-b12 loop around the FAD-bound molecule were clearly observed for evaluation, whereas the electron density maps of these loops in AhSIP-B were disordered (Supporting Fig. S2). DaliLite server was used to identify the structural differences of AhSIP with homologous proteins. It was found that molecule A of AhSIP has structural similarity with Shewanella putrefaciens SIP (PDB entry 2GPJ, named SpSIP, z-score: 29.3), Shewanella frigidimarina SIP (6GEH/SfSIP/27.8), and Thermobifida fusca SIP (4YHB/ TfFscN/26.6). The overall architecture of the SIPs consisting of FAD and NAD(P)H binding domains are similar, but these two domains show different conformations with an r.m.s. deviation of 1.18e2.2 Å (Supporting Fig. S3). Moreover, AhSIP has low sequence identities with SpSIP (34%), SfSIP (28%), and TfSIP (28%) (Supporting Fig. S4), which led to a unique substrate and siderophore binding pocket (see below).

3.2. FAD binding cavity of AhSIP In SIPs, FAD is essential for catalyzing NAD(P)H-dependent iron reduction [23,24]. The electron density map of FAD as a cofactor was clearly observed on the deep cleft between the FAD binding

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domain and NAD(P)H binding domain (Fig. 1A and Supporting Fig. S5A). The temperature factors of FAD of AhSIP-A and AhSIP-B were 17.0 Å2 and 33.0 Å2, respectively. When compared with the average temperature factor of all the atoms in the protein, the FAD in molecule A tightly interacted with AhSIP, whereas the high Bfactor of FAD in AhSIP-B was considered a loose interaction or low occupancy of FAD. In AhSIP-A, the isoalloxazine ring of FAD was inserted at the boundary between the FAD- and the NADH-binding domains, whereas the other parts of the FAD molecule laid at the interface between the FAD-binding domain and the connecting domain (Fig. 1B). FAD is surrounded by 25 amino acids (Supporting Fig. S5B); hydrogen bonds or salt bridges are formed with nine amino acids (Thr84, Asp98, Ala100, Ala108, Ser109, His102, Asp104, Glu255, Arg81) (Fig. 2A), and hydrophobic interactions are formed with sixteen amino acids (Thr146, Phe99, Gly105, Trp249, Gly252, Pro107, Asp254, Lys253, Arg250, His251, Gly106, Arg262, Phe83, His46, Thr82, Tyr248) (Fig. 2B). The isoalloxazine ring is stabilized through hydrogen bonds with the N atom of Thr84 (3.21 Å), O atom of Asp98 (2.72 Å) and N atom of Ala100 (2.72 Å) (Fig. 2A). The oxygen atoms of the diphosphate group of FAD are also stabilized through hydrogen bonds with the NE2 atom of His102 (3.30 Å), the N atom of Ala108 (3.11 Å), and the N atom of Ser109 (2.88 Å) of AhSIP; one of the oxygen atoms of phosphate group 2 forms hydrogen bonds with the N atom of Glu255 (2.98 Å) (Fig. 2A and Supporting Fig. S5B). In addition, the NE and NH1 atoms of Arg81 form interactions with phosphate group 1 and one interaction with phosphate group 2 of FAD (Fig. 2A). The catalytic function of FAD is concentrated in the isoalloxazine ring of flavin [25]. In AhSIP, FAD is also stabilized through hydrophobic interactions, and the isoalloxazine ring is stabilized through aromatic stacking interactions with His46, Phe83, and Tyr248 (Fig. 2B). In addition, an aromatic stacking arrangement maintains the adenine moiety between Trp249 and His102, and hydrogen bonding interactions between the protein and the 20 -hydroxyl of the ribose of FAD further reinforces the protein-cofactor interaction (Fig. 2A and B). Although FAD in AhSIP-B has a distinguished B-factor value versus AhSIP-A, the overall interaction between AhSIP-B and FAD is similar to that of FAD with AhSIP-A (data not show). In the SIP superfamily, the FAD domain has a highly conserved flavin binding amino acid sequence motif RXY(T/S) [13]. In contrast, AhSIP places Phe83 at the position corresponding to the tyrosine residue of RXY(T/S) (Fig. 2C and Supporting Fig. S4). Although Phe83 of AhSIP was not conserved with the tyrosine amino acid of the other SIPs, the pep interaction with flavin remained the same (Fig. 2D). The structural comparison of the FAD binding cavity in AhSIP and its homologous proteins showed that the FAD cofactor was confined by hydrophobic interactions in a typical conformation, as shown by other examples from this superfamily (Fig. 2D).

Fig. 1. Crystal structure of AhSIP. (A) Cartoon representation of one monomer of AhSIP illustrating the N-terminal NADH binding domain and C-terminal FAD binding domain. The a-helices, b-strands, and loops are colored red, yellow, and green, respectively, and the bound FAD cofactor is shown as a stick model. (B) Two molecules (chain A in blue, chain B in yellow) present in the asymmetric unit shown in surface format with the FAD cofactors in stick representation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 2. The FAD binding site of AhSIP. (A) FAD bound to the cavity is in yellow stick representation, and the residues are colored as gray sticks. Black dashed lines indicate hydrogen bonds or salt bridges. (B) Sphere representation of FAD surrounded by hydrophobic interactions with the amino acids shown as pink sticks. (C) Structure and sequence comparison of the FAD binding motif (RXY(T/S)) from AhSIP, SpSIP, SfSIP and TfSIP. FAD is represented as pink spheres. (D) Close-up of FAD and comparison of the stacking residues from AhSIP, SpSIP, SfSIP and TfSIP. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.3. Analysis of siderophore binding sites of SIPs Previous biochemical experiments on E. coli SIP suggest that the ferric-siderophore binding pocket is composed of a triad of basic amino acid residues [24]. Based on the comparative analysis of the amino acid sequence and crystal structure, the siderophore binding site of AhSIP is found at the interface of the two domains, consisting of the three basic amino acid residues including Lys48 of the FAD binding domain and His259 and Arg262 in the NADH binding domain (Fig. 3A). The side chains of these three basic amino acids are oriented toward the pocket. The siderophore binding pocket is approximately 16.5 Å away from the potential NADH binding site, and NADH as an electron donor is expected to reduce ferricsiderophore in subgroup I SIPs [10]. However, the siderophore binding pocket of AhSIP differs from the previously reported crystal structures of SpSIP and SfSIP (Fig. 3B and C). In SpSIP and SfSIP, the triad of basic amino acids that bind the ferric-siderophore are composed of lysine. In SpSIP, the side chains of the three lysine residues are oriented toward the inside of the ferric-siderophore pocket as in AhSIP. On the other hand, in SfSIP the side chain of Lys257 is directed outward from the siderophore binding pocket. In addition, other lysine residues in the triad of SpSIP and SfSIP show slight differences in their conformations (Fig. 3B and C). As a result, AhSIP Lys48 is conserved with other subgroup I SIPs, whereas His259 and Arg262 in AhSIP are replaced with lysine residues in other subgroup I SIPs. The basic triad of amino acids can have various conformations before the siderophore binds in SIPs. 3.4. Putative NADH binding sites of AhSIP SIPs can be divided into two subfamilies (group I and group II)

[26,27]. Group I SIPs, such as TfFscN and SpSIP, have a longer Cterminal a-helix containing 15e20 amino acids [10,28]. Group II has a relatively shorter C-terminus, such as in E. coli YqjH [24]. In addition, two subfamilies of SIPs are distinguished according to their C-terminal sequence alterations and have two different kinds of NAD(P)H binding motifs [(E(L)VL-X3-GE) and (TA-X3-E(L)VL-X3GE)], where X is any residue. Sequence analysis of AhSIP suggests that AhSIP contains the prominent C-terminal a-helix belonging to the group I subfamily and has the highly conserved NADH motif (E(L)VL-X3-GE) (Supporting Fig. S4). To date, no crystal structure has been reported for SIPs in complex with NAD(P)H; thus, the binding mechanism of NADH to SIP is still unclear. In previous studies, Sus Scrofa cytochrome b5 reductase was used as a model to understand the NADH binding site of the SIP family [28]. This protein consists of similar functional domains and performs the identical molecular mechanism as the SIPs with NADH and FAD [29]. Through comparative studies, the NAD(P)H binding site of TfFcsN was predicted to be composed of Thr119, Ala120, Glu173, Val174, Val221 and Phe222 [28]. However, our structural analysis shows that the residues Glu173 and Val174 lie near the NADH binding site, while Thr119, Ala120, Val221 and Phe222 lie in an unrelated position (Supporting Fig. S6A). Thus, we investigated putative NADH binding sites of AhSIP using a comparative study with cytochrome b5 reductase in complex with NADH and FAD (PDB entry: 3W2G) (Fig. 4 and Fig. S6B). Although AhSIP exhibits the low sequence identity of 20% with this cytochrome b5 reductase, the overall structure containing FAD and NADH domains shows similarity with a r.m.s. deviation of 2.468 Å (Fig. 4A). In the cytochrome b5 reductase, the FAD and NADH molecules interact with the FAD and NADH binding domains, respectively (Fig. 4B). The isoalloxazine ring of FAD and the

Fig. 3. Siderophore binding cavity of AhSIP and comparison its with homologs. Siderophore cavity and the triad of basic amino acid residues of (A) AhSIP (Lys48, His259, Arg262), (B) SpSIP (Lys45, Lys236 and Lys239) and (C) SfSIP (Lys49, Lys257, Lys259).

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Fig. 4. Putative NADH binding sites of AhSIP. (A) Superimposition of the SIP bound with FAD (blue color) and the cytochrome b5 reductase bound with FAD and NADH (gray color). (B) A close view of the FAD (yellow) of AhSIP and FAD (pink) and NADH (green) of the cytochrome b5 reductase are shown as sticks. The red dotted line indicates the configuration of the adenine moiety of the FAD of AhSIP and cytochrome b5 reductase. NADH binding mode of cytochrome b5 reductase (C) and putative NADH binding sites of AhSIP (D). Residues involved in NADH binding are presented with stick model and labeled. (E) The putative NADH binding site is occupied by the b10-a6 and b12-a8 loops, which are shown in cartoon with red color. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

nicotinamide moiety of NADH were 3.2 Å apart (Fig. 4B). The binding conformations of the isoalloxazine ring and phosphate groups of FAD are similar between this cytochrome b5 reductase and AhSIP, while the configuration of the adenine moiety of the FAD in cytochrome b5 reductase is directed to the solvent (Fig. 4B). In this cytochrome b5 reductase, the nicotinamide mononucleotide of NADH lies between the two domains, while the adenosine nucleotide of NADH surrounds the upper portion of the NADH domain (Fig. 4B). In this cytochrome b5 reductase, NADH was stabilized by hydrogen bonding (with Lys82, Tyr84, Thr153, Gln182, Asp211 and Phe272), hydrophobic interactions (Cys245 and Pro247) and a p-p interaction (Phe223) (Fig. 4C). The residues Lys82, Tyr84, Thr153, Gln182, Asp211, Cys245, Pro247 and Phe272 in cytochrome b5 reductase recognize NADH (Fig. 4C), whereas Ala100, His102, Thr146, Pro,171 Gly192, Pro199, Gly219, Glu221 and Val246 in AhSIP are located at the same structural positions as those mentioned for the cytochrome b5 reductase (Fig. 4D). As a result, the NADP-binding residues of cytochrome b5 reductase are not conserved with AhSIP. Moreover, in AhSIP, the putative NADH binding site is occupied by the b10-a6 and b12-a8 loops (Fig. 4E), suggesting that these loops undergo a conformational change for NADH binding during the activation process. Further experiments are required to clarify whether our predicted NADH binding cavity is accurate and elucidate the relationship with protein function. Acknowledgements The authors acknowledge the staff of the Pohang Light Source (PLS, Pohang, Republic of Korea) for their assistance with data collection. This work was supported by the Program for Liaoning Excellent Talents in University (grant no. LJQ2015030 to Y. Xu), the Fundamental Research Funds for the Central Universities (grant no. DC201502020203 to Y. Xu and grant no. DC201502020201 to C. Quan), and a National Research Foundation of Korea grant funded

by the Korean government (MOE) (Grant 2017R1D1A1B03033087 and 2017M3A9F6029736).

No.

NRF-

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.085. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.085. Conflicts of interest The authors have no conflicts of interest to report. References [1] G. Porcheron, A. Garenaux, J. Proulx, M. Sabri, C.M. Dozois, Iron, copper, zinc, and manganese transport and regulation in pathogenic Enterobacteria: correlations between strains, site of infection and the relative importance of the different metal transport systems for virulence, Front. Cell Infect. Microbiol. 3 (2013) 90. [2] D.J. Ferraro, A. Okerlund, E. Brown, S. Ramaswamy, One enzyme, many reactions: structural basis for the various reactions catalyzed by naphthalene 1,2-dioxygenase, IUCrJ 4 (2017) 648e656. [3] M.G. Repetto, N.F. Ferrarotti, A. Boveris, The involvement of transition metal ions on iron-dependent lipid peroxidation, Arch. Toxicol. 84 (2010) 255e262. [4] E.L. MacKenzie, K. Iwasaki, Y. Tsuji, Intracellular iron transport and storage: from molecular mechanisms to health implications, Antioxidants Redox Signal. 10 (2008) 997e1030. [5] E. Ahmed, S.J. Holmstrom, Siderophores in environmental research: roles and applications, Microb. Biotechnol. 7 (2014) 196e208. [6] B.R. Wilson, A.R. Bogdan, M. Miyazawa, K. Hashimoto, Y. Tsuji, Siderophores in iron metabolism: from mechanism to therapy potential, Trends Mol. Med. 22 (2016) 1077e1090. [7] Y. Han, K. Zang, C. Liu, Y. Li, Q. Ma, The putative siderophore-interacting protein from Vibrio anguillarum: protein production, analysis, crystallization

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