OhyA functional domains

S. aureus OhyA is a prototypical member of Family 11 in the fatty acid hydratase database (18). OhyA crystallizes with three protomers in the asymmetric unit (Fig. S1A), and the X-ray data collection and refinement statistics for all structures are listed in Table 1. Two protomers interact with each other to form the dimer, and the third protomer interacts with a symmetry-related protomer around a crystallographic 2-fold axis to form a dimer with a protomer in the adjacent asymmetric unit. OhyA dimerizes along the 2-fold noncrystallographic Q symmetry axis (Fig. 2A). Each protomer has a surface area of ~25,000 Å², and ~2500 Å² (10%) is primarily buried by the interacting helices α1, α3, and α4 in the amino terminal region that form the dimerization interface (Fig. 2A). All OhyA proteins are dimers (15, 20, 21, 22), except for the Rhodococcus erythropolis OhyA ortholog, which is a monomeric enzyme that lacks the key α1 dimerization helix (34).

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Figure 2

OhyA is a dimer composed of three functional domains. The fatty acid lobes are green, FAD lobes are yellow, and carboxy termini are blue. A, OhyA proteins use interacting helices α1, α3, and α4 to form the OhyA dimer interface along the Q axis. B, The OhyA protomer is divided into three functional domains: FAD lobe, fatty acid lobe, and carboxy terminus. C, The interior cavity of an OhyA protomer is mapped in solid color showing the contributions of the fatty acid (green) and FAD (yellow) lobes. Fatty acids enter the hydrophobic tunnel just below the carboxy terminus and FAD enters through the FAD lid at the opposite end. The active site is located at the intersection of the fatty acid and FAD cavities. D, The functional domains are mapped onto the primary sequence. The locations of key α helices, the sequence the covers FAD (Lid), and the active site loop (Loop) are indicated. FAD, flavin adenine dinucleotide; OhyA, oleate hydratase.

The S. aureus OhyA protomer has a bilobed structure for binding two substrates and a peripheral carboxy-terminal helical domain with an overall structure that is the same as reported for other OhyAs (Fig. 2B). Volkov et al. (21) divides the protein into four structural elements (I-IV; Fig. S1B); however, here we use a functional classification to describe the OhyA protomer domains (Fig. 2C). The FAD lobe contains the conserved dinucleotide binding motif/phosphate (FAD) binding signature sequence, the active site Loop, and the Lid covering the FAD entrance (Fig. S2). The FAD-binding domain (Fig. 2B) is a variant of the Rossmann fold consisting of a parallel five-stranded β-sheet flanked by three α-helices on one side and an additional crossover connection consisting of an antiparallel three-stranded β-sheet and α-helix on the other side (Fig. S1C). This topology is also observed in the FAD-containing glutathione reductase family (35) and corresponds to structural element I (Fig. S1C). The fatty acid lobe (Fig. 2B) creates a hydrophobic tunnel with an entrance just below the carboxy terminal domain that extends to the active site (Fig. 2C) and is constructed from structural elements II and III (Fig. S1D). The internal cavity in the FAD lobe merges with the fatty acid tunnel to form the active site at the domain interface (Fig. 2C). The carboxy terminal domain consists of three α-helices (Fig. 2B) and constitutes the structural element IV (Fig. S1B). The amphipathic helices have hydrophobic side chains facing inward to form a hydrophobic surface cavity and the polar side chains extend into the solvent. Abundant lysine and arginine residues give this region a positively charged electrostatic surface. The OhyA lobes are encoded in a nonlinear fashion using crossover connections to piece the lobe elements together to form the functional domain (Fig. 2D). The amino terminal element of the FAD lobe contains the dimerization helices (α1, α3, and α4), the sequence of 12 amino acids that covers the FAD binding site (Lid), and the conserved RGGREM active site Loop (Fig. 2D). The fatty acid lobe encodes helices α5 and α17 that form the entrance to the substrate tunnel, and helix α7 contributes the α-helix dipole to the active site. These highly conserved structural features do not change in our crystal structures, except for the Lid and the Loop sequences which undergo significant conformational changes depending on the bound ligands.

Fatty acid binding

Glu82 in the active site Loop (RGGREM) is critical for activity (Fig. 1C), and we mutated this residue to slow catalysis and capture complexes that were not obtained with the wild-type enzyme. The OhyA(E82A) structure in the absence of ligands (Protein Data Bank [PDB]: 7KAX) reveals the state of the empty fatty acid tunnel (Fig. 3A). The catalytic Loop residue Arg81 stands upright to create a wall at the end of the tunnel preventing the fatty acid from entering the active site. The OhyA(E82A)•oleate complex structure was obtained by soaking OhyA(E82A) crystals with oleate delivered with bovine serum albumin (PDB: 7KAY). Helices α5 and α17 form the tunnel entrance that is lined with hydrophobic amino acids that guide oleate into the tunnel (Fig. 3B). The complete electron density for oleate shows the fatty acid tail at the tunnel entrance with the carboxyl group leading the fatty acid toward the active site (Fig. 3B). Each of the protomers in the asymmetric unit of OhyA(E82A)•oleate complex (PDB: 7KAY) shows oleate in a slightly different location as it advances down the binding tunnel (Fig. S3). In MolA, there is a water molecule between the fatty acid carboxylate and Arg81, and the fatty acid tail extends to the tunnel entrance (Fig. 3C). In MolB, the fatty acid carboxylate moves closer to Arg81 forming an oleate–water–Arg81 hydrogen bond network (Fig. 3D). In MolC, the intervening water is absent, and the carboxylate forms direct hydrogen bond interactions with Arg81 (Fig. 3E). Oleate is blocked from entering the active site by the sidechain of Arg81. These data establish the location of the substrate binding tunnel in OhyA and the orientation of oleate as it approaches the active site.

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Figure 3

OhyA is a fatty acid binding protein. The transparent interior cavity surfaces are formed by the FAD (yellow) and fatty acid (green) lobes. A, View of the empty tunnel in the OhyA(E82A) structure (PDB: 7KAX). The Arg81 side chain walls off the end of the substrate binding tunnel at the entrance to the active site (red circle). B, The mouth of the tunnel is created by hydrophobic residues on helices α5 and α17. Oleate (black) sits in the substrate binding tunnel with its tail at the tunnel mouth in OhyA(E82A)•oleate complex (PDB: 7KAY, MolA). The oleate electron density calculated from a 2mF_O − DF_C map contoured at 1 σ (blue mesh) shows the carboxyl group pointing toward the bottom of the tunnel. C, The OhyA(E82A)•oleate complex (PDB: 7KAY, MolA) shows the fatty acid tail at the tunnel entrance and a water molecule (red sphere) between oleate and Arg81. D, In OhyA(E82A)•oleate complex (PDB: 7KAY, MolB) oleate is further into the tunnel forming an oleate–water–Arg81 hydrogen bond network. E, The water is absent from the tunnel, and oleate makes direct hydrogen bond contacts with Arg81 in OhyA(E82A)•oleate complex (PDB: 7KAY, MolC). Arrows denote the positions of the Δ9 double bond. FAD, flavin adenine dinucleotide; OhyA, oleate hydratase.

OhyA•FAD interactions

FAD is a nucleotide cofactor composed of riboflavin (isoalloxazine ring attached to a ribose) linked to adenosine diphosphate (Fig. 4A). The isoalloxazine moiety is a tricyclic molecule made up of a hydrophobic xylene ring and a polar uracil ring that are separated by a pyrimidine ring. FAD was soaked into crystals of OhyA•PEG400 to achieve the OhyA•PEG400•FAD ternary complex (PDB: 7KAW). The details of FAD–protein interactions with the catalytic Loop and active site Lid are best visualized in the ternary complex with the active site Lid closed over the FAD (Fig. 4A). The xylene ring faces a hydrophobic wall formed by residues contributed by the fatty acid lobe and is stabilized by a π–π stacking interaction with Phe506 and a cation–π interaction (36) with the Arg81 guanidinium group on the catalytic Loop. The uracil nitrogen atom N3 and carbonyl oxygen on carbon atom C4 form hydrogen bond connections to the backbone carbonyl and amide of Glu82, respectively (Fig. 4A). The carbonyl oxygen on carbon atom C2 also forms a hydrogen bond with the Thr508 backbone amide. The adenine moiety rests in a leucine and valine–rich hydrophobic cavity. Adenine nitrogen atoms N1 and N10 form hydrogen bond interactions with the Val250 backbone amide and carbonyl, respectively, and adenine nitrogen atom N3 forms a hydrogen bond interaction with the Glu57 backbone amide (Fig. 4A). Nucleotide binding proteins often stabilize pyrophosphate binding using a positive helix dipole (37). In OhyA, the FAD pyrophosphate moiety is stabilized by the positive dipole of helix α2 (Fig. S4) and hydrogen bonds with water molecules. The residues that make up helix α2 are located within the dinucleotide-binding motif/phosphate-binding signature sequence found in all glutathione reductase structural family members (35) (Fig. S2). The only direct hydrogen bond contact between the Lid and FAD is the backbone amide of Lid residue Ser64 interacting with a FAD phosphate (Fig. 4A). The weak stabilization of the closed Lid conformation explains why electron density for the Lid is most often not observed in OhyA structures.

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Figure 4

Organization of the active site by FAD binding.A, Schematic of hydrogen bond (purple lines), π–π stacking (green line), and cation–π (red line) interactions between OhyA structural elements and FAD in the OhyA•PEG400•FAD complex with the lid closed (PDB: 7KAW, MolC) created with Maestro. The locations of the catalytic Loop and Lid are highlighted (beige shading). The hydrophobic surface accommodating the FAD xylene ring is derived from the fatty acid lobe (green shading). B, The active site before FAD binding in the OhyA•PEG400 complex (PDB: 7KAV, MolA). The active site is formed by the FAD (yellow) and fatty acid (green) lobes and contains the substrate analog PEG400 (black) and water molecules (red spheres). The arrow denotes direction of the incoming fatty acid. C, The active site with FAD bound in the OhyA•PEG400•FAD complex (PDB: 7KAW, MolB). Same orientation and color coding as Panel B but containing FAD (cyan). D, Glu82 (magenta) interacts with four ordered waters in the OhyA•PEG400 complex (PDB: 7KAV, MolA). E, Glu82 (yellow) coordinates a single water following FAD (cyan) binding in the OhyA•PEG400•FAD complex (PDB: 7KAW, MolB). Dashed lines are hydrogen bonds connecting FAD to the backbone carbonyl and amide of Glu82. F, Overlay of Panels D and E illustrating the desolvation of the active site, the conformational change in the orientation of Glu82, and the substrate water. FAD, flavin adenine dinucleotide; OhyA, oleate hydratase; PEG400, polyethylene glycol 400.

The OhyA•PEG400 active site (PDB: 7KAV, MolA) in the absence of FAD is exposed to solvent and filled with water molecules (Fig. 4B). FAD binding in the OhyA•PEG400•FAD complex (PDB: 7KAW, MolB) squeezes the water molecules out of the active site with the exception of the substrate water (Fig. 4C). Glu82 on the catalytic Loop coordinates the substrate water from below the PEG400 substrate analog in the absence of FAD and also forms hydrogen bonds with three additional nearby water molecules in the active site (Fig. 4D). When FAD binds, the catalytic Loop conformation is altered by its direct interaction with FAD, and the Glu82 sidechain is repositioned to form hydrogen bonds with the substrate water (Fig. 4E). An overlay of these two states shows how FAD displaces the nearby water molecules and rotates Glu82 into a catalytic active conformation connected to a single substrate water molecule (Fig. 4F).

The active site lid

The FAD entrance to the OhyA active site is located at the interface of the FAD and fatty acid lobes along the P axis of the protein (Fig. 5A). In this view, the empty active site of the OhyA•PEG400 complex (PDB: 7KAW, MolA) is a gaping solvent-exposed hole in the protein surface, and the Lid that covers the active site is not observed. FAD binds into this cavity, but the Lid remains unstructured in OhyA•PEG400•FAD complex (PDB: 7KAW, MolB) (Fig. 5B). In OhyA•PEG400•FAD complex (PDB: 7KAW, MolC), FAD is bound in the pocket, and the Lid has undergone a conformational change from its flexible state to a 3₁₀-helix that covers the hole (Fig. 5C). Lid closure compresses the active site reducing the distance between Glu82 and PEG400 by >1 Å (Fig. S5). This compression means that the OhyA•PEG400•FAD complex with the Lid closed (PDB: 7KAW, MolC) does not contain the substrate water, but rather Glu82 is now directly hydrogen bonded to the backbone carbonyl of Met186 on the α7 helix dipole (Fig. S5B). The E. meningoseptica OhyA•PEG400•FAD structure with the Lid closed also does not contain a substrate water molecule in the active site (20). These observations suggest that Lid closure compresses the active site, shortening the distances between the substrate water, Glu82, and the oleate alkene to potentially accelerate catalysis.

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Figure 5

A flexible lid covers the active site.A, Exterior surface rendering of the OhyA•PEG400 complex (PDB: 7KAW, MolA) showing the open lid and the vacant FAD binding cavity exposed to solvent. FAD lobe is yellow, fatty acid lobe is green. OhyA protomers are rotated 90° about the P axis from the view in Figure 2B. B, FAD (cyan) bound at the active site in the OhyA•PEG400•FAD complex with the FAD lid open (PDB: 7KAW, MolB). C, The Lid residues (cyan) form a 3₁₀ helix that closes over the active site in the OhyA•PEG400•FAD complex (PDB: 7KAW, MolC). FAD, flavin adenine dinucleotide; OhyA, oleate hydratase; PEG400, polyethylene glycol 400.

Fatty acid trajectory through the active site

Comparison of the positions of PEG400, oleate, and h18:0 product shows that PEG400 is a nonreactive substrate analog that adopts the same curled conformation in the active site as the substrate and product (Fig. 6). The PEG400 substrate analog and oleate substrate positions are nearly identical while FAD remains in the same position and maintains the same backbone hydrogen bond interactions with the catalytic Loop across all substrate and product complexes (Fig. 6, A and B). Oleoyl alcohol is an OhyA substrate (23) and the position of the terminal hydroxyl of PEG400 is a model for the location of the oleoyl alcohol hydroxyl or oleate carboxyl just before catalysis occurs. The OhyA•product•FAD complex (Fig. 6C) shows that the hydroxy-fatty acid product is also located in the same place except it has rotated further around the active site. These data indicate that the terminal carboxyl of oleate enters the active site to participate in hydronium ion formation through formation of hydrogen bonds with Glu82 as observed in the OhyA•PEG400•FAD complex (see below).

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Figure 6

Substrate, substrate analog, and product complexes. The substrate tunnel and active site surfaces are derived from the FAD (yellow) and fatty acid (green) lobes. A, Positions of PEG400 (black) and FAD (cyan) in the active site of the OhyA•PEG400•FAD complex (PDB: 7KAW, MolA). PEG400 curls over FAD which forms two hydrogen bonds with the backbone carbonyl and amide of Glu82 on the catalytic loop (yellow). B, Oleate (black) is released from the tunnel and curls into the active site over FAD (cyan) which has the same hydrogen bond interaction with the catalytic loop (yellow) in the OhyA(E82A)•oleate•FAD complex (PDB: 7KAZ, MolB). Arrow denotes the position of the Δ9 double bond. C, Positions of the 10-hydroxystearate (h18:0) product (black) in relation to FAD (cyan) and the catalytic loop (yellow) in the OhyA(E82A)•h18:0•FAD product complex (PDB: 7KAZ, MolC). FAD, flavin adenine dinucleotide; OhyA, oleate hydratase; PEG400, polyethylene glycol 400; h18:0, (R)-10-hydroxyoctadecanoic acid.

The wall created by Arg81 between the tunnel and the active site prevents oleate from entering the active site in the OhyA(E82A)•oleate complex (Fig. 7A). The catalytic water molecule is present and coordinated by a helix dipole from α7 that ends in the backbone carbonyl of Met186 (Fig. 7A). The direction of the helix dipole focuses a partial negative charge on the substrate water, which stably coordinates the water even though Glu82 is missing from the active site (Fig. S4). FAD binding engages the backbone of the catalytic Loop and flips Arg81 out of the tunnel allowing oleate to rotate into the active site (Fig. 7B). The FAD-induced conformational change creates a continuous surface creating a donut-shaped active site. The h18:0 product has advanced ~4 to 5 Å into the active site and curls around the doughnut shaped cavity (Fig. 7C). It remains unclear how the h18:0 exits the protein. The orientation of the product suggests it may exit by moving up the fatty acid tunnel. However, when FAD leaves the active site, there is a large opening that the more polar hydroxylated product may diffuse out.

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Figure 7

Progression of oleate into the active site. Oleate is black, active site loop residues are yellow, and FAD is cyan. The active site surface is derived from the FAD (yellow) and fatty acid (green) lobes. The α7 helix dipole is colored from blue (amino terminus) to red (carboxy terminus). A, The OhyA(E82A)•oleate complex (PDB: 7KAZ, MolA) shows the α7 helix dipole interacting with the water substrate (red sphere). B, The OhyA(E82A)•oleate•FAD complex (PDB: 7KAZ, MolB) shows FAD interactions with the catalytic loop rotates Arg81 out of the tunnel, and the oleate is released to curl into the active site. The substrate water molecule is anchored by the α7 helix dipole and oleate makes van der Walls contact with the FAD ring system. C, The OhyA(E82A)•h18:0•FAD product complex (PDB: 7KAZ, MolC) shows the substrate water transferred to the 10-carbon of the substrate and the acyl chain curled further into the active site above the FAD ring system. D, Oleate is prevented from entering the active site by Arg81 that blocks the tunnel and forms direct hydrogen bond contact with the oleate carboxyl group. E, Arg81 rotates out of the tunnel to allow the oleate to move into the active site. F, The product complex shows the catalytic loop is in a different conformation with Arg78 (brown) adjacent to the fatty acid carboxyl and Arg81 is rotated to point away from the fatty acid. FAD, flavin adenine dinucleotide; OhyA, oleate hydratase; PEG400, polyethylene glycol 400; h18:0, (R)-10-hydroxyoctadecanoic acid.

Movements of the catalytic loop

The Loop contains two arginine residues (Arg78 and Arg81) whose movements direct the fatty acid around the active site doughnut. Before FAD binding, Arg81 walls off the fatty acid in the substrate tunnel from access to the active site as the Arg81 side chain hydrogen bonds with the oleate carboxylate (Fig. 7D). Arg78 is pointed away from the oleate. FAD binding engages the catalytic Loop backbone (Fig. 6) triggering the Arg81 side chain to shift 8 Å from its upright conformation releasing the fatty acid into the active site (Fig. 7E). In this new conformation, the Arg81 side chain is still hydrogen bonded to the fatty acid carboxylate pulling the acyl chain into the active site torus (Fig. 7E). The Arg78 side chain is still oriented away from the active site and does not interact with oleate substrate (Fig. 7D). However, in the h18:0 product complex, the catalytic Loop arginine residues flip positions (Fig. 7F). Arg78 side chain swivels 11 Å toward h18:0 and the Arg81 side chain turns an additional 8 Å and now points away from the active site. These conformational changes place Arg78 behind the fatty acid carboxyl to facilitate the continued rotation of the product around the active site. These data reveal the subtle changes in the catalytic Loop residues that guide the oleate through the active site as the reaction proceeds.

Role of Tyr201

Mutagenesis experiments suggested a conserved tyrosine (Tyr201 in OhyA) has a role in catalysis and was proposed to house the proton abstracted by the substrate cis double bond (20). Tyr201 is in the fatty acid lobe and the tyrosyl oxygen forms a 2.7 Å hydrogen bond interaction with the backbone carbonyl of Val505 on the FAD lobe to seal one side of the active site (Fig. 8A). The almost contiguous electron density across Tyr201 and Val505 in all structures reflects the strength of this interlobe intramolecular hydrogen bond. This Val505 interaction fixes the position of a structured loop that makes a π–π stacking interaction between the Phe506 and the FAD xylene ring and a hydrogen bond between the Thr508 backbone amine and the FAD uracil ring (Fig. 4A). These structural roles for Tyr201 account for the lower thermal stability of OhyA(Y201F) (Fig. 1B). The Tyr201–Val505 hydrogen bond connection is intact after oleate enters the active site (Fig. 8B) and is observed in all OhyA structural complexes (Fig. 8) making the Tyr201 hydroxyl proton unavailable to participate in catalysis. This means Tyr201 does not donate a proton to carbon 9 of oleate to initiate the OhyA reaction. Rather than participate in catalysis, the Tyr201 hydroxyl forms a hydrogen bond network that stabilizes the hydroxy-fatty acid product (Fig. 8C). The h18:0 hydroxy participates in a Phe187–h18:0–Tyr201–Val505 hydrogen bond network (Fig. 8C). Water addition to the carbocation intermediate generates a product that would exactly fit into this network. OhyA production is stereoselective, and we determined if this selectivity was altered in the OhyA(Y201F) mutant. Mass spectrometry confirmed carbon-10 is the position for hydroxylation for OhyA and OhyA(Y201F) (Fig. S6). Product stereochemistry was determined by derivatization and chiral chromatography (Fig. S7), and (R)-10-hydroxyoctadecanoic acid was the sole product in both OhyA and OhyA(Y210 F). Loss of the Tyr201 tyrosyl oxygen in OhyA(Y201F) disrupts the active site hydrogen bond network that stabilizes the OhyA active site structure and the hydroxy-fatty acid product.

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Figure 8

Role of Tyr201 and the hydronium ion. Contributions from the FAD lobe are yellow, fatty acid lobe is green, fatty acid is black, and FAD is cyan. A, The OhyA(E82A)•oleate complex (PDB: 7KAZ, MolA) shows the α7 helix dipole colored from blue (amino terminus) to red (carboxy terminus). Tyr201 on the fatty acid lobe makes a 2.7 Å hydrogen bond interaction with the carbonyl of Val505 on the FAD lobe to seal one side of the active site. B, The OhyA(E82A)•oleate•FAD complex (PDB: 7KAZ, MolB) shows the oleate chain rotating into the active site above FAD making van der Waals contact with the hydrophobic xylene ring. C, The OhyA(E82A)•h18:0•FAD product complex (PDB: 7KAZ, MolC) shows the Phe187 backbone carbonyl-product hydroxyl-Tyr201 hydroxyl-Val505 backbone carbonyl hydrogen bond network that stabilizes water addition to carbon-10 of the fatty acid. D, Two rotated snapshots of the coordination network show three direct interactions with contiguous electron density to the catalytic water molecule (red sphere). An interaction between Glu82 and the PEG400 terminal hydroxyl is evident. Electron density calculated from a 2mF_O − DF_C map contoured at 1 σ (blue mesh). E, Diagram of the hydrogen bonding network surrounding the substrate water. Distances represent measurements between oxygen atoms are shown with three hydrogen bonds connected to the water oxygen indicating it is a positively charged hydronium ion. FAD, flavin adenine dinucleotide; OhyA, oleate hydratase; PEG400, polyethylene glycol 400; h18:0, (R)-10-hydroxyoctadecanoic acid.

Cloning, expression, and purification of OhyAs

The S. aureus ohyA DNA sequence was synthesized and cloned into pET28a with an N-terminal His tag for purification from Escherichia coli as previously described (15) to form pOhyA. The OhyA(E82A) mutation was introduced by modifying pOhyA using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies, Cat# 210519) and primers OhyA(E82A)-F (5'-CCGCGGTGGTCGTGCAATGGAGAACCACT-3') and OhyA(E82A)-R (5'-AGTGGTTCTCCATTGCACGACCACCGCGG-3') to form pOhyA(E82A). The OhyA(Y201F) mutation was introduced by modifying pOhyA using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies, Cat# 210519) and primers OhyA(Y201F)-F (5'-TCTGCAATGGAAATGCGTCGCTTTCTAATGCGATTC-3') and OhyA(Y201F)-R (5'-GAATCGCATTAGAAAGCGACGCATTTCCATTGCAGA-3') to form pOhyA(Y201F). The pOhyA, pOhyA(E82A), and pOhyA(Y201F) plasmids were transformed into E. coli BL21(DE3) cells (Millipore Sigma, Cat# 70,235), and isolates were obtained on Luria broth agar imbedded with 50 μg/μl kanamycin (Gold Biotechnology, Cat# K-120). Transformants were amplified in Luria broth containing 50 μg/μl kanamycin (Gold Biotechnology, Cat#K-120) and shaken at 37 °C, 200 rpm. Cells were grown to an OD₆₀₀ of 0.6 and then cooled to 16 °C before induction with 1 mM isopropyl-β-D-thiogalactoside (Gold Biotechnology, Cat# I2481 C) overnight. Cells were harvested and then lysed in buffer containing 20 mM Tris, pH 8.0, 10 mM imidazole, 200 mM NaCl, and a dissolved tablet of Pierce protease inhibitor (Thermo Fisher Scientific, Cat# A32955). The OhyA, OhyA(E82A), and OhyA(Y201F) proteins were separated from cell lysates by nickel agarose beads (Gold Biotechnology, Cat# H-320) and eluted in buffer containing 20 mM Tris, pH 8.0, 250 mM imidazole, and 200 mM NaCl. The eluant was gel filtered into buffer containing 20 mM Tris, pH 8.0, 200 mM NaCl using the HiLoad Superdex 200 column (Cytiva Life Sciences, Cat# 28989335) with dimensions 16 mm x 60 cm. The molecular weights of the OhyA, OhyA(E82A), and OhyA(Y201F) proteins were estimated using an XBridge BEH SEC 200 Å 3.5 μm column (Waters Corporation, Cat# 176003595) with dimensions 7.8 mm x 150 mm. The standard curve for the XBridge BEH SEC column used thyroglobulin (669 kDa), immunoglobin (150 kDa), BSA (66.4 kDa), and myoglobulin (17 kDa).

OhyA activity assay

The OhyA, OhyA(E82A), and OhyA(Y201F) activities were assayed as described previously (15). Briefly, 20 μl reactions containing 50 mM potassium phosphate buffer, pH 6.0, 150 mM NaCl, 10 mM DTT, 50 μM FAD (Millipore Sigma, Cat# F6625), 0.2 mg/ml fatty acid-free bovine serum albumin (Millipore Sigma, Cat# A6003), 20 μM [1-¹⁴C]oleate (specific activity 54.3 mCi/mmol) (PerkinElmer, Cat# NEC317250UC), and 1 μg OhyA, 100 μg OhyA(E82A), or 10 μg OhyA(Y201F) were incubated at 37 °C for 20 min. These enzyme concentrations were experimentally determined based on prior reactions with varying enzyme concentration monitoring detectable hydroxy fatty acid product formation. A 10 μl aliquot of the reactions were spotted on a Silica Gel H thin-layer plate (Spectrum Chemical, Cat# 476–10,450) and separated using chloroform:methanol (90:10, v/v). Hydroxy fatty acid product formation was visualized using a Typhoon PhosphorImager and quantified using ImageQuant (Cytiva Life Sciences, https://www.cytivalifesciences.com/en/us/shop/protein-analysis/molecular-imaging-for-proteins/imaging-software).

OhyA product stereochemistry

OhyA and OhyA(Y201F) activity assays were performed with oleate (Matreya, LLC, Cat# 1022), the fatty acids extracted and the solvent removed under nitrogen (42). The position of hydroxylation was determined by product scans operating a Sciex QTrap 4500 mass spectrometer in the negative mode and compared with (R)-9-hydroxyoctadecanoic acid (Larodan Research Grade Lipids, Cat# 14–1809) and (R/S)-10-hydroxyoctadecanoic acid (AA Blocks, LLC, Cat# AA0032L4) standards as described previously (15). The source parameters were ion spray voltage, −4500 V; curtain gas, 15 p.s.i.; temperature, 250 °C; collision gas, high; ion source gas 1, 15 p.s.i.; ion source gas 2, 20 p.s.i.; declustering potential, −25 V; and collision energy, −35 V.

The enantiomer composition was determined by derivatization and chiral chromatography. The (R/S)-10-hydroxyoctadecanoic acid (15 mg, 0.047 mmol, 1 eq) (AA Blocks, LLC, Cat# AA0032L4) was combined with HBTU (O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) coupling reagent (21 mg, 0.056 mmol, 1.2 eq) (Oakwood Chemical, Cat# 009019) and placed under a nitrogen atmosphere. The reaction was diluted with 0.250 ml of tetrahydrofuran (Millipore Sigma, Cat# 401757) then treated with (R)-α-methylbenzylamine (6.5 μl, 0.051 mmol, 1.1 eq) (Millipore Sigma, Cat# 115541) followed by diisopropylethylamine (12 μl, 0.070 mmol, 1.5 eq) (Millipore Sigma, Cat# D125806) at room temperature. The reaction was stirred overnight then diluted with 0.5 ml of methanol and eluted through a short plug of SiliaBond-Carbonate (0.5 g) (Silicycle, Cat# R60430B) stacked onto a short plug of SiliaBond-Tosic Acid (0.5 g) (Silicycle, Cat# R66030B) with the aid of an additional 3 ml of methanol by gravity. The collected filtrate was concentrated, and the resulting white solid was dissolved in methanol at 2 mg/ml suitable for chiral chromatography. Fatty acids from the enzyme reaction were derivatized using the same procedure and injected directly onto the column after solvent switch from tetrahydrofuran (Millipore Sigma, Cat# 401757) to methanol. Chiral chromatography was performed on a Waters UPC2 supercritical fluid chromatography system on a Chiral Technologies Chiralpak IG (4.6 mm x 150 mm) column (Chiral Technologies, Cat# 87,324) using an isocratic 30% methanol/supercritical carbon dioxide mobile phase. Analyte peaks appeared at 6.2 (R) and 6.6 min (S).

Fatty acid association

The OhyA protein was assayed to determine fatty acid binding ability using tritium labeled oleic acid. Briefly, 100 μl reactions containing 50 mM potassium phosphate buffer, pH 6.0, 150 mM NaCl, 1% v/v dimethyl sulfoxide, 20 μM OhyA, and 1 μM [9,10-³H]oleate (specific activity 54.0 Ci/mmol) (PerkinElmer, Cat# NET289001MC) were mixed and incubated at 37 °C for 1 h. A 50 μl aliquot of the reaction was separated using an XBridge BEH SEC 200 Å 3.5 μm column (Waters Corporation, Cat# 176003595) with dimensions 7.8 mm x 150 mm. The elution was collected in 20 s increments, and the fractions were analyzed by scintillation counting.

Thermal stability

The nanoDSF Prometheus NT.48 was used to directly measure dynamic light scattering of OhyA, OhyA(E82A), and OhyA(Y201F). Briefly, 20 μl samples containing 50 mM potassium phosphate buffer, pH 6.0, 150 mM NaCl and 10 μM OhyA, OhyA(E82A), or OhyA(Y201F) were mixed and loaded into Prometheus NT.48 Series nanoDSF Grade High Sensitivity Capillaries (NanoTemper Technologies, Cat# PR-C006) by capillary force action. The capillaries were heated from 20 °C to 90 °C at a rate of 1 °C/min, and the UV light scattering was recorded to determine the apparent aggregation and T_agg.

Crystallization and structure determination

The OhyA protein was concentrated to 20 mg/ml for crystallization. Initial screening was performed at 20 °C against the Protein Complex Suite (NeXtal Biotechnologies, Cat# 130715) by hanging drop vapor diffusion method combining 200 nl protein and 200 nl precipitant. Diffraction quality crystals were obtained by combining 1.5 μl protein and 1.5 μl 20% v/v PEG400, 100 mM calcium acetate, and 100 mM MES, pH 6.0. Crystals were cryo-protected with 20% v/v PEG400, 100 mM calcium acetate, 100 mM MES, pH 6.0, and 30% glycerol and then flash-frozen in liquid nitrogen for X-ray diffraction experiments. OhyA was solved by the molecular replacement method using the program Phaser (43) and the coordinates of E. meningoseptica OhyA (PDB: 4UIR) (20) as the search model. These crystals contained the OhyA•PEG400 binary complex. The OhyA•PEG400•FAD ternary complex was achieved by soaking OhyA•PEG400 crystals in 20 μl 20% v/v PEG400, 100 mM calcium acetate, 100 mM MES, pH 6.0, and 750 μM FAD (Millipore Sigma, Cat# F6625) overnight at room temperature. Crystals were cryo-protected in 20% v/v PEG400, 100 mM calcium acetate, 100 mM MES, pH 6.0, and 30% glycerol and then flash-frozen in liquid nitrogen. Attempts to achieve the OhyA•FAD binary or OhyA•PEG400•FAD ternary complex by co-crystallization failed because of FAD precipitation. Attempts to deliver fatty acid in the crystallization drop using common agents that solubilize hydrophobic compounds including detergents and dimethyl sulfoxide failed. The OhyA(E82A)•oleate binary complex was achieved by incubating 7 mg/ml (100 μM) OhyA(E82A) with 1 mg/ml fatty acid-free bovine serum albumin (Millipore Sigma, Cat# A6003) and 500 μM oleate (Matreya, LLC, Cat# 1022) for 3 days in 150 mM sodium chloride and 50 mM potassium phosphate, pH 6.0 at room temperature prior to crystallization. The mixture was screened at 20 °C against the PACT Suite (NeXtal Biotechnologies, Cat# 130718) by hanging drop vapor diffusion method combining 200 nl protein and 200 nl precipitant. Diffraction quality crystals were obtained by combining 1.5 μl protein and 1.5 μl 15% v/v PEG3000, 200 mM magnesium chloride, and 100 mM sodium cacodylate, pH 6.5. Crystals were cryoprotected with 15% v/v PEG3000, 200 mM magnesium chloride, 100 mM sodium cacodylate, pH 6.5, and 30% glycerol and then flash-frozen in liquid nitrogen. The OhyA(E82A) substrate-free structure was solved using the same method but omitting oleate. The OhyA(E82A)•oleate•FAD ternary complex was achieved soaking crystals containing OhyA(E82A)•oleate in 20 μl 15% v/v PEG3000, 200 mM magnesium chloride, 100 mM sodium cacodylate, pH 6.5, and 750 μM FAD (Millipore Sigma, Cat# F6625) overnight at room temperature. Crystals were cryoprotected with 15% v/v PEG3000, 200 mM magnesium chloride, 100 mM sodium cacodylate, pH 6.5, and 30% glycerol, flash-frozen again in liquid nitrogen, and X-ray diffraction data were re-collected. Crystals containing OhyA•PEG400 and OhyA(E82A)•oleate that were soaked with FAD turned bright yellow, which is consistent with incorporation of the oxidized cofactor. All diffraction datasets were collected at the SER-CAT beamline 22-ID at the Advanced Photon Source and processed using KYLIN (44) and HKL2000 (45). The structures of the OhyA•PEG400•FAD ternary complex, OhyA(E82A)•oleate binary complex, and OhyA(E82A)•oleate•FAD ternary complex were solved by molecular replacement using the OhyA•PEG400 binary complex structure as a search model. The structures were completed by iterative rounds of refinement using phenix.refine (46) and manual rebuilding using Coot (47). The refinement was monitored by following the R_free value calculated from a random subset (5%) of omitted reflections. A summary of the data processing and structure refinement statistics is provided in Table 1. The figures related to protein structures were generated with PyMOL (48).

In-silico analyses

Surface areas were calculated in PyMOL (48) as previously described (49). The buried surface area by dimerization was calculated by subtracting the surface area of two protomers linked along the Q axis from the combined surface area of the two individual protomers.

We thank Pam Jackson for molecular biology assistance. We also thank the St Jude Structural Biology X-ray Center for crystallography support, Sivaraja Vaithiyalingam (Molecular Interaction Analysis Shared Resource, St Jude Children's Research Hospital) for sample analysis by nano-DSF, and the Hartwell Center DNA Sequencing Shared Resource for DNA sequencing.