Structure and reaction mechanism of a novel enone reductase Feng Hou1, Takuya Miyakawa1, Nahoko Kitamura2, Michiki Takeuchi2, Si-Bum Park2, Shigenobu Kishino2, Jun Ogawa2 and Masaru Tanokura1 1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan 2 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan

Keywords crystal structure; enone reductase; fatty acid; NADH oxidase/flavin reductase family; reaction mechanism Correspondence M. Tanokura, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 1138657, Japan Fax: +81 3 5841 8023 Tel: +81 3 5841 5165 E-mail: [email protected] (Received 23 October 2014, revised 28 January 2015, accepted 16 February 2015) doi:10.1111/febs.13239

Recently, a novel gut-bacterial fatty acid metabolism, saturation of polyunsaturated fatty acid, that modifies fatty acid composition of the host and is expected to improve our health by altering lipid metabolism related to the onset of metabolic syndrome, was discovered in Lactobacillus plantarum AKU 1009a. Enzymes constituting the pathway catalyze sequential reactions of free fatty acids without CoA or acyl carrier protein. Among these enzymes, CLA-ER was identified as an enone reductase that can saturate the C=C bond in the 10-oxo-trans-11-octadecenoic acid (KetoB) to produce 10-oxo-octadecanoic acid (KetoC). This enzyme is the sole member of the NADH oxidase/flavin reductase family that has been identified to exert an enone reduction activity. Here, we report both the structure of holo CLAER with cofactor FMN and the KetoC-bound structure, which elucidate the structural basis of enone group recognition of free fatty acids and provide the unique catalytic mechanism as an enone reductase in the NADH oxidase/flavin reductase family. A ‘cap’ structure of CLA-ER underwent a large conformational change upon KetoC binding. The resulting binding site adopts a sandglass shape and is positively charged at one side, which is suitable to recognize a fatty acid molecule with enone group. Based on the crystal structures and enzymatic activities of several mutants, we identified C51, F126 and Y101 as the critical residues for the reaction and proposed an alternative electron transfer pathway of CLA-ER. These findings expand our understanding of the complexity of fatty acid metabolism. Database The atomic coordinates have been deposited in the Protein Data Bank (PDB), www.pdb.org (PDB ID 4QLX, 4QLY)

Introduction Fatty acids serve many essential functions in living organisms, including central roles in biological energy storage (e.g. triacylglycerols and cholesteryl esters), structural integrity and dynamics (phospholipids, plasmalogens and sphingolipids) and the control of cellular metabolism and cell physiology (diacylglycerols, phos-

phatidylinositols and eicosanoids) [1,2]. Fatty acids can be remodeled in many ways, such as by elongation, insertion or removal of double bonds, or covalent binding to proteins. A number of these modification pathways have been identified and the roles of the enzymes involved in them have been investigated [3–5].

Abbreviations ACP, acyl carrier protein; ACS, acyl-coenzyme A synthetase; FabI, an enone-acyl reductase; KetoB, 10-oxo-trans-11-octadecenoic acid; KetoC, 10-oxo-octadecanoic acid; WT, wild-type.

FEBS Journal (2015) ª 2015 FEBS

1

F. Hou et al.

Novel enzyme in NADH oxidase/flavin reductase family

As a common initial step of the metabolic processes, the acyl-coenzyme A synthetases (ACSs) catalyze a reaction in which the fatty acid is bound to CoA and converted to the ‘activation’ form [6]. The acyl-CoA is directly used in the subsequent metabolic process or converted to the acyl carrier protein (ACP) bound form. Recently, a novel pathway of fatty acid metabolism, the saturation of polyunsaturated fatty acids, which generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids and partially saturated fatty acids as intermediates, was discovered in Lactobacillus plantarum AKU 1009a [7]. The action of the gut-bacterial fatty acid saturation metabolism modifies the fatty acid composition of the host and is expected to improve human health by altering lipid metabolism, which is related to the onset of metabolic syndrome [7]. Interestingly, the enzymes involved in the gut-bacterial fatty acid saturation metabolism catalyze modification reactions of free fatty acids without CoA or ACP. In the fatty acid saturation pathway, CLA-ER was identified as an enone reductase and shown to saturate the C=C double bond of the 10-oxo-trans-11-octadecenoic acid (KetoB) to 10-oxo-octadecanoic acid (KetoC), a key step of linoleic acid saturation to oleic acid and trans-10-octadecenoic acid (Fig. 1) [7]. Enone reductase is widely used for the stereoselective reduction of alkenes. In particular, the members of the old yellow enzyme family have been extensively studied and used in the asymmetric reduction of various alkenes [8]. Old yellow enzymes are identified in various pathways such as enoyl-CoA reductase in fatty acid biosynthesis and morphinone reductase in morphine biosynthesis [9] but catalyze the reactions with the same catalytic residue, tyrosine, as proton donor [8]. Other enone group reducing enzymes with different

structures and catalytic mechanisms have also been identified. FabI, an enone-acyl reductase involved in bacterial type II fatty acid biosynthesis, reduces the enone group carried by ACP using cofactor NADH. This enzyme is a member of the short-chain dehydrogenase/reductase superfamily, the members of which are characterized by a catalytic triad of tyrosine, lysine and serine residues [10]. A reduction reaction using an oxyanion hole has been proposed for LovC, which was identified as a trans-acting enone reductase in lovastatin biosynthesis and possesses a medium-chain dehydrogenase/reductase fold [11]. On the other hand, CLA-ER belongs to the NADH oxidase/flavin reductase family. Enzymes belonging to this family have been shown to perform several functions. The majority of the family members catalyze the reduction of nitro compounds through a Ping Pong Bi Bi mechanism in which the first step reduces flavin by transferring two electrons from NAD(P)H [12,13]. Enzymes in this family are also reported to have other activities, such as iodotyrosine deiodination activity [14] and flavin fragmentation activity [15]. However, CLA-ER is the sole member of the NADH oxidase/flavin reductase family that has been shown to exert an enone-reducing activity. Here, we report the crystal structures of CLA-ER in both the holoenzyme state with FMN and the KetoCbound state. These structures clarified the mechanism involved in the recognition of free fatty acids with an enone group and revealed the catalytic mechanism underlying the enone-reducing activity of members of the NADH oxidase/FMN reductase family. These results expand our understanding of the complex mechanisms of fatty acid metabolism.

Results Overall structures of CLA-ER

Fig. 1. Schematic diagram of the enone reduction catalyzed by CLA-ER. The enone group of KetoB and the corresponding part of KetoC are highlighted in red.

2

The crystal structure of holo CLA-ER was determined
resolution. CLA-ER forms a homodimeric at 2.10 A structure (Fig. 2A), which is consistent with the results of size exclusion chromatography (Fig. 2B). In the FMN-bound structure, each CLA-ER molecule adopts an a + b fold composed of nine a-helices and five bstrands, and is divided into two subdomains termed the core subdomain and flexible subdomain (Fig. 2A). The core subdomain possesses a central b-sheet that is formed by four anti-parallel b-strands (b1‒b4) together with a fifth b-strand on the C-terminal tail of the other protomer (b50 ). The central b-sheet is surrounded by six a-helices (a1‒4, a7 and a8) and a short a-helix on the C-terminal tail of the other protomer (a90 ) FEBS Journal (2015) ª 2015 FEBS

F. Hou et al.

Fig. 2. Crystal structure of holo CLA-ER. (A) Ribbon diagram of the dimeric structure of CLA-ER with FMN. One protomer is colored in grey and the other protomer is color-coded from blue (Nterminus) to red (C-terminus). The FMN molecule is shown as a magenta stick. (B) Size exclusion chromatography of CLA-ER. The curves of holo CLA-ER with FMN and its KetoC-bound state are colored in red and blue, respectively. (C) Hydrophobic residues on the dimer interface of the a7helix. (D) Cleft for FMN binding. The two protomers, A and B, are colored in grey and pink, respectively. Residues forming the cleft are highlighted by green sticks. Protomer codes are labeled in parentheses. Water molecules are indicated by cyan spheres. The FMN molecule is shown as a slate-colored stick–ball model.

Novel enzyme in NADH oxidase/flavin reductase family

A

B

C

D

(Fig. 2A). These observations suggest that the domain swapping of the C-terminal tails contributes to stabilization of the dimeric structure of CLA-ER. In addition, two protomers contact each other through tight hydrophobic interactions between the a7 helices (Fig. 2C), and two FMN molecules are located within clefts formed at the interface of the two core subdomains (Fig. 2A,D). The flexible subdomain adopts a helix–loop–helix structure (residues 97–132) with a rel
2) compared with that of atively high B-factor (47.6 A
2). In the dimeric structure, the core subdomain (28.7 A two flexible subdomains are located at both sides of the core subdomains (Fig. 2A). The crystal structure of CLA-ER in the presence of
resolution (Fig. 3A). KetoC was determined at 2.15 A KetoC-bound CLA-ER showed a structure similar to that of CLA-ER in the KetoC-free state, except for the orientation of the flexible subdomains with a low
2) and C-terminal
2; overall, 23.1 A B-factor (34.6 A tails (Fig. 3B). According to the electron density, the C-terminal tails of CLA-ER extended away from the core subdomain and interacted with other CLA-ER dimers in a manner similar to that in the structure of holo CLA-ER (Fig. 3B,C). The results of the size exclusion chromatography showed that CLA-ER also FEBS Journal (2015) ª 2015 FEBS

formed a dimer in the KetoC-bound state, and the molecular size was a little more compact than in the KetoC-free state (Fig. 2B), which indicates that the extension of the C-terminal tails was due to the conditions of crystallization. Therefore, a modified dimeric structure with C-terminal tail swapping was adopted in this study (Fig. 3A). The electron density of KetoC was observed near the FMN molecule bound into the clefts between the two protomers (Fig. 3D). Cofactor binding in the clefts of the dimer interface The FMN molecules bound to the dimer interface through extensive interactions with two protomers both in the holo and KetoC-bound states of CLA-ER (Figs 2A and 3A). The isoalloxazine rings of the FMN molecules have a bending conformation, indicating that they are in the reduced form (Fig. 3B). One protomer provides direct hydrogen bonds with FMN and the other forms hydrophobic interactions and watermediated hydrogen bonds with FMN (Figs 3D and 4A). Several critical interactions between CLA-ER and FMN are well conserved for enzymatic function of the NADH oxidase/flavin reductase family. The N1‒

3

Novel enzyme in NADH oxidase/flavin reductase family

A

D

Fig. 3. Crystal structure of CLA-ER in the presence of KetoC. (A) Ribbon diagram of the modified dimeric structure of CLA-ER with both FMN and KetoC. The two protomers, A and B, are colored the same as those in (B). FMN molecules are shown as slate-colored stick models. The KetoC molecules are indicated by yellow sphere models. (B) Molecular contacts of CLA-ER in crystal. Three molecules of the CLA-ER dimer are shown in different colors. (C) Comparison of the C-terminus between the KetoC-free structure (magenta) and KetoC-bound structure (orange). (D) Electron density map of KetoC and hydrogen bonding to FMN. The Fo  Fc electron density map of KetoC is shown as a grey mesh contoured at 1.0r. FMN and KetoC molecules are shown as slate and yellow stick models, respectively. Residues forming hydrogen bonds with FMN are shown as a wheat-colored stick model, and protomer codes are labeled in parentheses. Hydrogen bonds are indicated by red dashed lines.

B

C

A

F. Hou et al.

B

Fig. 4. LIGPLOT+ diagrams for non-covalent interactions of CLA-ER with FMN (A) or KetoC (B).

C2 = O2 locus of FMN interacts with Arg24 (Figs 3D and 4A), which is thought to be relevant to stabilizing the anionic form of the reduced FMN and increasing 4

its redox potential. On the other hand, FMN N5 directly participates in the substrate dehydrogenation and interacts with the backbone of Ser168 on the FEBS Journal (2015) ª 2015 FEBS

F. Hou et al.

Novel enzyme in NADH oxidase/flavin reductase family

A

B

D C

Fig. 5. Conformational changes for KetoC recognition. (A) Structural superimposition of holo CLA-ER with FMN (pink) and CLA-ER in complex with FMN and KetoC (slate). The FMN and KetoC molecules are represented by sticks and spheres, respectively. (B) KetoC-binding site in CLA-ER. The electrostatic surface potential of the binding site is color-coded from red (negative charge) to blue (positive charge). Residues around the enone group and FMN are represented as green stick–ball models. (C) Residues forming the KetoC-binding site. Residues belonging to the ‘cap’ structure are represented by a slate-colored stick-and-ball model. The KetoC molecule is shown as a yellow sphere. Protomer codes are labeled in parentheses. (D) Conformational changes of F126 and Y101. The structures of holo CLA-ER with FMN and its further complex with KetoC are colored in pink and slate, respectively. The KetoC molecule is presented as yellow sticks.

si-face of FMN (Figs 3D and 4A). This hydrogen bond is expected to increase the oxidative power of FMN [16]. With these interactions, flavins are buried in the surface of the clefts mentioned above and their re-faces are oriented toward solvents (Fig. 2D). Recognition mechanism of KetoC with a large conformational change Superposition of the structures of CLA-ER with and without KetoC revealed that the core subdomains had
for almost the same conformation (rmsd of 0.425 A 171 Ca atoms). However, a significant conformational change was observed in the flexible subdomain. Upon KetoC binding, the flexible subdomain rotates approximately 45° towards the core subdomain of the other protomer in the dimer (Fig. 5A) and acts as a ‘cap’ to capture KetoC. The conformational change of the flexible subdomain causes CLA-ER to change from an open to a closed form (Fig. 5A), resulting in the formation of a binding site for KetoC recognition (Fig. 5B). The binding site adopts a long sandglass shape that is narrow in the middle and relatively wide at both ends (Fig. 5B). FEBS Journal (2015) ª 2015 FEBS

The residues for KetoC recognition come from the two protomers (Fig. 5C), which indicates that the dimeric structure of CLA-ER is essential for the KetoC recognition. C51, F126 and the flavin ring of FMN are located in the middle region of the binding site and provide a suitable space to recognize the carbonyl group, Ca atom and Cb atom of KetoC (Figs 1 and 5B), which corresponds to the position of the enone group of KetoB. The chemical structures of KetoB and KetoC reveal the high degree of similarity between these two compounds (Fig. 1). The carbon atoms at the Ca and Cb positions form a double bond
in KetoB but a single with a bond length of 1.34 A
in KetoC. The bond bond with a length of 1.54 A angles are 121 and 110 in KetoB C–C=C and KetoC C–C–C, respectively. These differences allow us to consider that KetoB forms almost the same structure as KetoC in the crystal structure of CLA-ER in complex with FMN and KetoC. Among the residues involved in the formation of the binding site, two basic residues, R24 and R118, are located on one side of the binding site and the other side consists of only hydrophobic residues and two polar amino acids (Y130 and S168) (Fig. 5C). The distribution of these residues

5

Novel enzyme in NADH oxidase/flavin reductase family

A

B

C

results in a basic half and a hydrophobic half of the binding site, which is proper for recognition of the carboxy group side and alkyl group side, respectively (Fig. 5B). However, there is no residue forming a hydrogen bond or salt bridge with the carboxy group of KetoC (Figs 4B and 5C). The wide open spaces and lack of any hydrophilic interactions at the ends of the binding site lead to a loose recognition at both ends of KetoC, which is considered to be the reason for the weak electron density at both ends. The KetoC-bound structure also explains an ingenious recognition system of the enone group of substrate KetoB. In the structure, the carbonyl group of KetoC was recognized by the amino group of C51 and the hydroxy group at the C20 position of FMN through hydrogen bonds (Fig. 5D). The Ca and Cb atoms of KetoC, corresponding to the C=C group of the enone group of KetoB, were fixed by the flavin ring of FMN and the side chains of C51 and F126 (Fig. 5C,D). Superposed structures in the open and closed forms revealed that F126 underwent the inversion of its side chain followed by the conformational change of its flexible subdomain. The inverted side chain of F126 acts as a ‘lock’ to accurately fix the 6

F. Hou et al.

Fig. 6. Catalytic mechanism for enone reduction. (A) Active site of CLA-ER. The residues in the CLA-ER, FMN and KetoC molecules are presented as slate, green and yellow sticks, respectively. A water molecule is indicated by a cyan sphere. Protomer codes are labeled in parentheses. (B) Catalytic activities of CLA-ER and its mutants. (C) Proposed models of enone reduction by CLA-ER. The electron transfer pathway is indicated by black arrows. The enone group of KetoB, the corresponding parts in KetoC and the proposed intermediate are highlighted in red. The peptide bond between A50 and C51 is also highlighted in red.

spatial arrangement of the Ca and Cb atoms. An obvious movement of Y101 was also induced by the conformational change accompanying the inversion of
towards the active site to F126. Y101 moved 8.4 A
from the thiol group of C51 (Fig. 5D). locate at 3.3 A Structural basis for the catalytic activity of CLA-ER In the KetoC-bound structure, the N5 atom of the fla
from the Cb vin ring is located at the position 3.1 A atom (Fig. 6A), which is suitable for hydride anion transfer. Among the residues adjacent to the Ca, C51 is the sole hydrophilic residue. The Ca and Cb atoms correspond to the C=C group of the substrate KetoB. Thus, C51 may not only contribute to the spatial arrangement of the C=C group but may also play a key role in the protonation of KetoB (Figs 5B and
from C51 and 6A). Y101 locates at the position 3.3 A is considered to facilitate the protonation process together with C51. To identify the residues critical for the reaction, enzyme assays were performed with the wild-type (WT) CLA-ER and its F126, C51 and Y101 mutants. FEBS Journal (2015) ª 2015 FEBS

F. Hou et al.

The catalytic activity of CLA-ER was remarkably decreased by the F126A mutation, indicating that the ‘lock’ residue F126 is important for the catalytic activity of CLA-ER (Fig. 6B). Compared with the WT protein, the activity of the C51A mutant was decreased by approximately 60% (Fig. 6B). This result shows that C51 is not essential for the activity of CLA-ER but can facilitate the reaction. Interestingly, a mutation of C51 to a serine residue did not affect the catalytic activity, which suggests that a polar residue can improve the activity of CLA-ER and supports the notion that C51 is involved in the electron transfer pathway. On the other hand, Y101F mutation had the most crucial influence on the catalytic activity among the three residues (Fig. 6B). Y101 underwent a
from C51 remarkable movement and located at 3.3 A (Fig. 6A). Thus, Y101 occupied the space of F126 in the open state of CLA-ER, which indicates that it may not only be involved in the electron transfer pathway but may also contribute to the rearrangement of the side chain of F126 (Fig. 5D).

Discussion Catalytic mechanism for enone reduction Several catalytic mechanisms have been reported for the reduction of enone reductase using cofactor FMN. Old yellow enzymes are reported to utilize a tyrosine residue as proton donor [8] and acyl-CoA dehydrogenases catalyze the a,b-dehydrogenation using a glutamic acid residue [17]. However, based on the KetoC-bound structure of CLA-ER and the results of enzyme assays, we propose a different catalytic mechanism of CLAER from those reported previously (Fig. 6C). After the reduction of FMN in the first step of the Ping Pong Bi Bi mechanism of the NADH oxidase/flavin reductase family, the carbonyl group of substrate KetoB is recognized by the hydrogen bonds with the amino group of C51 and hydroxy group of FMNH, followed by further recognition of the C=C group by hydrophobic interaction with the side chain of F126. These recognitions result in relocation of the Cb atom to a site near the N5 position of FMNH and the Ca atom to a site near the thiol group of the C51 residue. The C=C group of KetoB was reduced by obtaining a hydride from the N5 position of FMNH and a proton from the hydroxy group of Y130 through the thiol group of C51 (Fig. 6C). The pKa of C51 in CLA-ER was calculated using H++ server [18]. The pKa value of C51 (9.5) is similar to the pKa of tyrosine residue in old yellow enzymes (around 9.1) [19], indicating that C51 is competent as a proton donor. However, enzyme FEBS Journal (2015) ª 2015 FEBS

Novel enzyme in NADH oxidase/flavin reductase family

assays showed that C51 was not an essential residue for the reaction, which implies that there is another pathway for the electron transfer (Fig. 6C). We propose that electrons can also be transferred to solvent water through the peptide bond between C51 and A50 (Fig. 6A,C). A similar electron transfer pathway through a peptide bond has also been proposed in the proton pumping in cytochrome c oxidase [20]. In the present case, the first step of the reaction could result in an enol intermediate, which could be converted to substrate by itself in the following step (Fig. 6C). Distinction of enone reductases in the NADH oxidase/flavin reductase family Although there are numerous enzymes belonging to the NADH oxidase/flavin reductase family, CLA-ER is the sole enzyme showing enone-reducing activity. Based on the structural findings of CLA-ER, we explain the reason for this novel activity in the NADH oxidase/flavin reductase family and propose a novel class that exerts activities similar to CLA-ER. The ‘cap’ structure of CLA-ER can be used as a guide for characterizing the enone-reducing activity, since it is essential for the recognition of KetoB. The DALI server was used to search for enzymes with structures similar to CLA-ER and with Z-scores above 11 (Fig. 7). A search for enzymes with different amino acid sequence identities above 40% was performed using BLAST (Figs 7A and 8). These sequential and structural homologous proteins were compared with CLA-ER. The ‘cap’ structure of CLA-ER was not conserved in any other enzymes. Here, we classify enzymes belonging to the NADH oxidase/flavin reductase family and having ‘cap’ structures similar to that of CLA-ER as the ER-like group. Other enzymes in this family possess only the central conserved structure that is related to the core subdomain of CLA-ER or other accessory structures in addition to the central conserved structure (Fig. 7B). Lack of the ‘cap’ structure is considered to be the reason that they do not show the enonereducing activity. As described in the reaction mechanism, C51, Y101 and F126 are especially important for the catalytic activity of CLA-ER (Figs 5D and 6B), and the water molecule, which is recognized by A50, S55, D141 and D145, may provide an alternative electron transfer pathway for the reaction. However, the ER-like group enzymes for which the structures were determined did not conserve the residues corresponding to A50, C51, S55, Y101, D141, D145 and F126 in CLA-ER (Fig. 8). Therefore, these residues in CLA-ER are considered to be the characteristic residues allowing this enzyme of the NADH oxidase/flavin

7

F. Hou et al.

Novel enzyme in NADH oxidase/flavin reductase family

A

B

Fig. 7. Comparison of CLA-ER with enzymes in the NADH oxidase/flavin reductase family. (A) Phylogenetic tree of CLA-ER and the enzymes with amino acid sequences or structures similar to CLA-ER. Enzymes with the reported structures are presented as the PDB code plus sequence identity (red) and Z-score in parentheses (blue) to CLA-ER. ND plus a sequence identity (red) represents an enzyme whose structure is not determined. The accession numbers of these enzymes are WP_003637313.1 (ND-96), WP_003662875.1 (ND-74), YP_003173365.1 (ND-70), WP_003639645.1 (ND-64), WP_003143577.1 (ND-58), WP_003648915.1 (ND-54), YP_003601906.1 (ND-49), YP_002886965.1 (ND-44) and WP_007718811.1 (ND-40). The enzymes are divided into two groups termed ‘ER-like’ and ‘others’. (B) Structural comparison of CLA-ER with typical enzymes belonging to the ‘others’ group. The two structures with the highest Z-scores (20) in comparison to CLA-ER in the ‘others’ group are presented as ribbon models. The PDB codes are presented under the individual structure. Conserved subdomains (core subdomains) are colored according to their groups in (A). Variable subdomains are highlighted in slate.

Fig. 8. Multiple sequence alignment of enzymes belonging to the ER-like group. Enzymes with the reported structures are presented as the PDB code plus the sequence identity to CLA-ER. ND plus a sequence identity represents an enzyme whose structure is not determined. Identical residues for reaction and interaction with water molecule are shaded in red and cyan, respectively.

reductase family to exert its enone-reducing activity. By means of the above-described BLAST search, we found an array of enzymes that conserve these 8

residues. Some of these enzymes (ND96, ND74, ND70 and ND64) have been identified in bacteria belonging to the Lactobacillus genus, which is widely distributed FEBS Journal (2015) ª 2015 FEBS

F. Hou et al.

in the human gastrointestinal tract. Therefore, the activity of ER-like enone reductases may have extensive effects on the fatty acid metabolism, especially the saturation of polyunsaturated fatty acids, in humans. CoA- or ACP-independent recognition of fatty acids The binding of CoA or ACP to fatty acids is considered to be helpful for promoting further modifications. These carriers not only allow for the easy transfer of fatty acids to other molecules, but also contribute to the recognition of reactive structures on acyl chains. In the structure of human mitochondrial 2,4-dienoyl-CoA reductase complexed with trans-2,trans-4-hexadienoylCoA, the CoA group forms several direct or water-mediated hydrogen bonds with side chains of the protein, which contributes to special arrangement of the dienoyl group for a saturation reaction [21]. Docking simulation of other enone-CoA reductases has also indicated that the CoA group interacts extensively with enzymes and contributes to substrate recognition [22,23]. On the other hand, a structural study of ACPbound FabI has revealed that the complex is primarily stabilized by the interactions between acidic residues of ACP and the substrate-binding loop of FabI. By these interactions, ACP is able to successfully deliver its substrate into the FabI active site through a minor entrance [10]. Unlike these enzymes, CLA-ER catalyzes the reduction reaction of free fatty acids with an enone group. It seems reasonable to suppose that fatty acids with a carbonyl group would have difficulty entering a narrow pocket and thus that the hydrophobic environment for fatty acid recognition is also not suitable for the entry of carbonyl groups. To recognize free fatty acids, CLA-ER adopts an open form in a fatty-acidfree state, which results in exposure of the cleft for the preliminary recognition of fatty acids (Fig. 2D). The open form of CLA-ER allows its substrate to approach the binding cleft without the help of a carrier. Additionally, the positive charges of the cleft electrostatically attract the carboxy group of fatty acid, which contributes to the orientation of the substrate (Fig. 5B). Further recognition depends on the hydrogen bonds of substrate with FMN and C51, and the large conformational change in the ‘cap’ structure. Therefore, both the charge distribution of the cleft and transformation of the open-closed structures make the substrate recognition possible without the combination with CoA or ACP. These features enable CLA-ER to reduce fatty acids that are difficult to link to CoA or FEBS Journal (2015) ª 2015 FEBS

Novel enzyme in NADH oxidase/flavin reductase family

ACP. The present study has clarified the mechanism for fatty acid recognition and binding, but the mechanism for the release of fatty acids is still not clear. Fatty acids may be released in some specific environment such as a lipid-rich environment, or transferred to following enzymes. Moreover, the sandglass shape of the catalytic site and open channel on both ends raise the possibility that CLA-ER recognizes the wide variety of fatty acids with an enone group and a long alkyl chain. Therefore, CLA-ER participates in the saturation reaction of the enone groups of various fatty acids in L. plantarum and/or enteral environments and provides bioactive metabolites of polyunsaturated fatty acids, i.e. hydroxy fatty acids, oxo fatty acids, conjugated fatty acids and partially saturated fatty acids, to host organisms through the saturation metabolism.

Experimental procedures DNA construction and cloning The gene encoding the full-length CLA-ER (Genbank accession no. NC_004567; region 60505-61350) was inserted into the pET-47b(+) vector from Novagen (Tokyo, Japan) between the SmaI and EcoRI sites to be produced as a recombinant protein with an N-terminal His6-tag sequence (MAHHHHHHSAALGVLFNGP). The plasmid was then used to produce a variety of mutations by using site-directed mutagenesis: the C51A, C51S, Y101F and F126A mutants. All mutations were constructed using the QuikChange site-directed mutagenesis kit with the list of primers (Table S1) and the expression vector for the WT protein as a template. The digestion with restriction enzyme DpnI was carried out at 37 °C for 1 h to remove the parent DNA, and the mutations were confirmed by a DNA sequencing service (FASMAC).

Protein expression and purification The protein was overexpressed using Escherichia coli Rosetta (DE3) cells from Novagen. The expression of the CLA-ER gene was induced by the addition of 0.5 mM IPTG at 298 K when the optical density at 600 nm reached 0.6. After culturing overnight, the cells were harvested by centrifugation at 5180 g for 10 min and frozen at 193 K. Purification was conducted at 277 K. The frozen cells were resuspended in buffer containing 20 mM Tris/HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 20 lM NADH and 10 lM FMN, and then disrupted by sonication. After centrifugation at 40 000 g for 30 min, the supernatant was applied onto an Ni-nitrilotriacetic acid superflow column from Qiagen (Tokyo, Japan). After washing with the same buffer, the fusion protein was

9

F. Hou et al.

Novel enzyme in NADH oxidase/flavin reductase family

cleaved on the column using HRV3C protease from Novagen at 277 K overnight. Two additional amino acids (GP) remained on the N terminus of the CLA-ER coding sequence. The cleaved protein was eluted with the same buffer and dialyzed against 20 mM Tris/HCl (pH 8.0) and 1 mM DTT. As the final purification step, anion-exchange chromatography was performed with a Resource Q 6 mL column from GE Healthcare (Tokyo, Japan). FMN and NADH were added to the purified protein to final concentrations of 2 mM and 4 mM, respectively. The protein solution was dialyzed against 20 mM Tris/HCl (pH 8.0) and then concentrated to 12 mgmL1. The concentration was measured by BCA assay [24].

Crystallization For the crystallization of CLA-ER with FMN, 4 mM NADH and 2 mM FMN were added to the protein solution. Initial crystallization trials were performed by the sitting-drop vapor-diffusion method in 96-well plates from Corning (Tokyo, Japan) using the sparse-matrix screening kits, Crystal Screen HT and Index HT from Hampton Research (Aliso Viejo, CA, USA), at 293 K. Drops containing equal volumes (0.2 lL) of protein solution and reservoir solution were equilibrated against a 40 lL reservoir solution. After optimization of the crystallization conditions, the crystals of holo CLA-ER with FMN grew under the condition of 0.1 M BisTris (pH 6.0) and 21% poly(ethylene glycol) monomethyl ether 5000. For crystallization of CLA-ER with both FMN and KetoC, 4 mM NADH, 2 mM FMN and 2 mM KetoC were added to the protein solution and the mixture was incubated at 4 °C overnight. The mixture was filtered with Cosmospin filter G (Nacalai Tesque, Kyoto, Japan) with a pore size of 0.2 lm for crystallization using the same methods as above. After optimization, the crystal of CLA-ER in complex with FMN and KetoC grew under the conditions of 100 mM sodium cacodylate (pH 6.8), 200 mM NaCl and 2.0 M ammonium sulfate.

Data collection, structure determination and refinement Crystals were picked up in a nylon loop (Hampton Research) and then flash-cooled using a nitrogen stream. X-ray diffraction data were collected at a wavelength of
on the AR-NE3A beamline at the Photon Fac1.0000 A tory (Tsukuba, Japan) using an ADSC Quantum 270 CCD detector. The diffraction data were indexed, integrated in space group P1 and C2221 with XDS and scaled with XSCALE [25], respectively. Upper resolution limits were assessed based on the balance of completeness, I/r(I) and CC-0.5. The crystals of holo CLA-ER with FMN and its further complex with KetoC contained four and two molecules in the asymmetric unit, respectively. The initial structure of

10

holo CLA-ER was determined by molecular replacement phasing with MOLREP using monomers of the nitroreductase enzyme from Enterobacter cloacae (PDB ID 1KQB) as a search model [26]. The new model was iteratively refined using PHENIX [27] and manually refurbished with COOT [28]. The final Rwork and Rfree values were 17.6% and 22.4%, respectively. The structure of CLA-ER in complex with FMN and KetoC was determined by molecular replaceTable 1. Data collection and refinement statistics.

Data collection X-ray source Wavelength (
A) Space group Unit-cell parameters (
A) Unit-cell parameters (°) Resolution range (
A)a No. observed reflectionsa No. unique reflectionsa Average redundancya Completeness (%)a Rsyma Rpim (%)a Average I/r(I)a CC-1/2 Refinement Rwork (%) Rfree (%) No. reflections No. atoms Protein Ligand Water Mean B value (
A2) Protein Ligand Water rms deviations Bond lengths (
A) Bond angles (°)

FMN-bound CLA-ER

CLA-ER complexed with FMN and KetoC

Photon Factory AR-NE3A 1.0000 P1 a = 49.7, b = 60.9, c = 72.6 a = 85.6, b = 98.9, c = 111.6 20.0–2.00 (2.06–2.00)

C2221 a = 64.9, b = 68.9, c = 197.5

20.0–1.95 (2.00–1.95)

194 897 (7476)

231 601 (15 025)

50 917 (3278)

32 720 (2386)

3.8 (2.3) 96.8 0.068 3.5 17.5 99.9

7.1 (6.3)

(84.2) (0.566) (36.5) (2.35) (77.6)

17.3 (28.1) 22.6 (33.7) 50 901

99.9 0.013 5.7 11.9 99.7

(100) (0.806) (101.1) (2.5) (87.1)

18.7 (24.9) 23.0 (30.9) 32 707

6624 124 358

3363 109 280

32.5 24.9 33.9

27.0 26.1 31.8

0.008 1.041

0.008 1.063

a

Values in parentheses are for the highest resolution shell. Rsym = Σhkl [(Σi |Ii  ‹I›|)/Σi|Ii|], where Ii is the ith intensity measurement of reflection hkl, including symmetry-related reflections, and ‹I› is its average. Rpim = Σhkl[1/(N  1)]1/2Σj|Ij  ‹I›|/Σhkl(ΣjIj), where N is the redundancy, Ij is the jth intensity measurement of reflection hkl, and ‹I› is its average. CC-1/2 is percentage of correlation between intensities from random half-datasets. The Rfree factor was calculated using 5% of the reflections omitted from the refinement.

FEBS Journal (2015) ª 2015 FEBS

F. Hou et al.

ment phasing using the structure of holo CLA-ER as the search model and refined using the same procedure as for holo CLA-ER. The final Rwork and Rfree values were 18.7% and 22.5%, respectively. All data processing and refinement statistics are summarized in Table 1.

Novel enzyme in NADH oxidase/flavin reductase family

Author contributions M.T. and J.O. designed research; F.H., T.M., N.H., M.K., S.P and S.K. performed research; F.H. and T.M. analyzed data; and F.H., T.M. and M.T. wrote the paper.

Sequence and structural analysis CLA-ER homologs were identified using BLAST [29]. Structurally similar enzymes were identified using DALI [30]. Multiple sequence alignment was performed using CLUSTALW [31]. The phylogenetic tree was drawn using EVOLVIEW (www.evolgenius.info/evolview). PYMOL was utilized for structural superimpositions and the calculation of electrostatic surface potential [32]. The interactions between CLAER and ligands were calculated and drawn using LIGPLOT+ [33].

Enzyme assays A reaction mixture containing 20 mM sodium citrate (pH 6.0), 1 mg KetoC dissolved in ethanol, 100 lM FMN, 10 mM NADH and 2 lM purified enzyme was incubated under microaerobic conditions in a sealed chamber with an O2 absorber from Anaeropack Kenki (Osaka, Japan) and Mitsubishi Gas Chemical (Tokyo, Japan) and gently shaken (200 r.p.m.) at 37 °C for 2 h. Lipid extraction was performed as described previously [7]. The extracted lipids were dissolved in 2 mL of methanol and 3 mL of benzene and then methylated with 150 lL of 1% diazomethane at room temperature for 30 min. The fatty acid methyl esters were concentrated by evaporation under reduced pressure. The resulting fatty acid methyl esters were dissolved in chloroform and analyzed by gas chromatography using a Shimadzu GC-1700 gas chromatograph fitted with a capillary column (SPB-1; 30 m length 9 0.25 mm internal diameter; Supelco). The initial column temperature was 180 °C for 30 min. The temperature was subsequently increased to 220 °C at a rate of 40 °Cmin1 and maintained for 24.5 min. The fatty acid peaks were identified by comparing retention times to known standards.

Acknowledgements We would like to thank the beamline staff at Photon Factory. The synchrotron radiation experiments were performed at AR-NE3A in the Photon Factory, Tsukuba, Japan (Proposal No. 2012G561). This work was supported by the Targeted Proteins Research Program (TPRP) of the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT), the Platform for Drug Discovery, Informatics, and Structural Life Science of MEXT, and the Bio-Oriented Technology Research Advancement Institution of Japan.

FEBS Journal (2015) ª 2015 FEBS

References 1 Tvrzicka E, Kremmyda LS, Stankova B & Zak A (2011) Fatty acids as biocompounds: their role in human metabolism, health and disease–a review. Part 1: classification, dietary sources and biological functions. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 155, 117–130. 2 Kremmyda LS, Tvrzicka E, Stankova B & Zak A (2011) Fatty acids as biocompounds: their role in human metabolism, health and disease: a review. part 2: fatty acid physiological roles and applications in human health and disease. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 155, 195–218. 3 Chan DI & Vogel HJ (2010) Current understanding of fatty acid biosynthesis and the acyl carrier protein. Biochem J 430, 1–19. 4 Schweizer E, Werkmeister K & Jain MK (1978) Fatty acid biosynthesis in yeast. Mol Cell Biochem 21, 95–107. 5 Kindl H (1993) Fatty acid degradation in plant peroxisomes: function and biosynthesis of the enzymes involved. Biochimie 75, 225–230. 6 Watkins PA (1997) Fatty acid activation. Prog Lipid Res 36, 55–83. 7 Kishino S, Takeuchi M, Park SB, Hirata A, Kitamura N, Kunisawa J, Kiyono H, Iwamoto R, Isobe Y, Arita M et al. (2013) Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition. Proc Natl Acad Sci USA 110, 17808–17813. 8 Stuermer R, Hauer B, Hall M & Faber K (2007) Asymmetric bioreduction of activated C=C bonds using enoate reductases from the old yellow enzyme family. Curr Opin Chem Biol 11, 203–213. 9 Barna T, Messiha HL, Petosa C, Bruce NC, Scrutton NS & Moody PC (2002) Crystal structure of bacterial morphinone reductase and properties of the C191A mutant enzyme. J Biol Chem 277, 30976–30983. 10 Rafi S, Novichenok P, Kolappan S, Zhang X, Stratton CF, Rawat R, Kisker C, Simmerling C & Tonge PJ (2006) Structure of acyl carrier protein bound to FabI, the FASII enoyl reductase from Escherichia coli. J Biol Chem 281, 39285–39293. 11 Ames BD, Nguyen C, Bruegger J, Smith P, Xu W, Ma S, Wong E, Wong S, Xie X, Li JW et al. (2012) Crystal structure and biochemical studies of the trans-acting polyketide enoyl reductase LovC from lovastatin biosynthesis. Proc Natl Acad Sci USA 109, 11144–11149.

11

F. Hou et al.

Novel enzyme in NADH oxidase/flavin reductase family

12 Parkinson GN, Skelly JV & Neidle S (2000) Crystal structure of FMN-dependent nitroreductase from Escherichia coli B: a prodrug-activating enzyme. J Med Chem 43, 3624–3631. 13 Race PR, Lovering AL, Green RM, Ossor A, White SA, Searle PF, Wrighton CJ & Hyde EI (2005) Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme. J Biol Chem 280, 13256–13264. 14 Thomas SR, McTamney PM, Adler JM, LarondeLeblanc N & Rokita SE (2009) Crystal structure of iodotyrosine deiodinase, a novel flavoprotein responsible for iodide salvage in thyroid glands. J Biol Chem 284, 19659–19667. 15 Taga ME, Larsen NA, Howard-Jones AR, Walsh CT & Walker GC (2007) BluB cannibalizes flavin to form the lower ligand of vitamin B12. Nature 446, 449–453. 16 Ghisla S & Massey V (1989) Mechanisms of flavoprotein-catalyzed reactions. Eur J Biochem 181, 1–17. 17 Ghisla S & Thorpe C (2004) Acyl-CoA dehydrogenases. A mechanistic overview. Eur J Biochem 271, 494–508. 18 Anandakrishnan R, Aguilar B & Onufriev AV (2012) H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res 40, W537–W541. 19 Meah Y & Massey V (2000) Old yellow enzyme: stepwise reduction of nitro-olefins and catalysis of acinitro tautomerization. Proc Natl Acad Sci USA 97, 10733–10738. 20 Yoshikawa S, Shinzawa-Itoh K, Nakashima R, Yaono R, Yamashita E, Inoue N, Yao M, Fei MJ, Libeu CP, Mizushima T et al. (1998) Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase. Science 280, 1723–1729. 21 Alphey MS, Yu W, Byres E, Li D & Hunter WN (2005) Structure and reactivity of human mitochondrial 2,4-dienoyl-CoA reductase: enzyme-ligand interactions in a distinctive short-chain reductase active site. J Biol Chem 280, 3068–3077. 22 Hu K, Zhao M, Zhang T, Zha M, Zhong C, Jiang Y & Ding J (2013) Structures of trans-2-enoyl-CoA reductases from Clostridium acetobutylicum and Treponema denticola: insights into the substrate specificity and the catalytic mechanism. Biochem J 449, 79–89.

12

23 Bond-Watts BB, Weeks AM & Chang MC (2012) Biochemical and structural characterization of the trans-enoyl-CoA reductase from Treponema denticola. Biochemistry 51, 6827–6837. 24 Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ & Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76–85. 25 Kabsch W (2010) Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr D Biol Crystallogr 66, 133–144. 26 Vagin A & Teplyakov A (2000) An approach to multicopy search in molecular replacement. Acta Crystallogr D Biol Crystallogr 56, 1622–1624. 27 Adams PD, Afonine PV, Bunk oczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213–221. 28 Emsley P & Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132. 29 Altschul SF, Madden TL, Sch€affer AA, Zhang J, Zhang Z, Miller W & Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402. 30 Holm L & Rosenstr€ om P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38, W545–W549. 31 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. 32 DeLano WL. The PyMOL Molecular Graphics System, http://www.pymol.org in. 33 Laskowski RA & Swindells MB (2011) LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 51, 2778–2786.

Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site: Table S1. Oligonucleotide primers used in the mutation studies.

FEBS Journal (2015) ª 2015 FEBS