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Structural analyses of the Group A flavin-dependent monooxygenase PieE reveal a sliding FAD cofactor conformation bridging OUT and IN conformations

Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen–oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD. These flavoenzymes play an important role in many biological processes, and their most distinct structural feature is the choreographed motions of flavin, which typically adopts two distinct conformations (OUT and IN) to fulfill its function. Notably, these enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate, but the molecular basis of this system remains elusive. Using protein crystallography, size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and small-angle X-ray scattering (SEC-SAXS) and activity assay, we report here a structural and biochemical characterization of PieE, a member of the Group A flavin-dependent monooxygenases involved in the biosynthesis of the antibiotic piericidin A1. This analysis revealed that PieE forms a unique hexamer. Moreover, we found, to the best of our knowledge for the first time, that in addition to the classical OUT and IN conformations, FAD possesses a “sliding” conformation that exists in between the OUT and IN conformations. This observation sheds light on the underlying mechanism of how the signal of substrate binding is transmitted to the FAD-binding site to efficiently initiate NADPH binding and FAD reduction. Our findings bridge a gap currently missing in the orchestrated order of chemical events catalyzed by this important class of enzymes.

Flavoenzymes catalyze an enormous range of chemical transformations that involve electron transfer processes.
The versatility of flavoenzymes can be attributed to multiple redox states of the tricyclic isoalloxazine ring of flavin cofactors FMN and FAD (1). Among the flavoenzymes, flavin-dependent monooxygenases catalyze the cleavage of the oxygen-oxygen bond of dioxygen followed by the incorporation of one oxygen atom into the substrate molecule (2). The reactive C4a-hydroperoxyflavin intermediate plays a central role in all of these flavin-dependent monooxygenases (3,4). Of note, a distinct feature of the widely studied Group A and Group B flavin-dependent monooxygenases is that they are single-component enzymes that use NAD(P)H as the external electron donor (5).
Most enzymes in Group A regioselectively catalyze the incorporation of hydroxyl at the ortho-or para-site of phenolic substrates (5). Previous studies of these enzymes suggest an electrophilic aromatic substitution mechanism in which the -OH terminal moiety of C4a-hydroperoxyflavin functions as an electrophile and the phenolic ring of substrates acts as a nucleophile (6). With the involvement of five components, including enzyme, substrate, FAD, NADPH, and O 2 , a typical catalytic cycle proposed for the Group A enzymes includes substrate recruitment, NADPH binding, FAD reduction, NADP ϩ release, oxygen binding and C4ahydroperoxyflavin formation, substrate hydroxylation, and oxidized FAD regeneration (3). The most distinct feature of these flavoenzymes is the choreographed motions of flavin, which typically adopts two distinct conformations (OUT and IN) to switch between the redox states to fulfill its function (7,8). Although the cofactor mobility is a prerequisite for the Group A enzymes, only a few members in this group have been observed through X-ray crystallography to possess two conformations (6) (Table S1). Notably, the Group A enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate (5), but its molecular basis remains elusive. Therefore, understanding the concerted interplay of substrate, NAD(P)H, and FAD within the enzyme active site will be of central importance to all of the Group A enzymes.
The prototype of the Group A flavin-dependent monooxygenases is para-hydroxybenzoate hydroxylase (PHBH), 2 on which extensive biochemical and structural characterizations have been carried out (9,10). This enzyme and its homologs are important for microbial degradation of aromatic compounds. Some Group A members are involved in human diseases. For example, kynurenine 3-monooxygenase (KMO) is a key enzyme in the metabolism of L-tryptophan and has been linked to neurodegenerative diseases such as Huntington's and Alzheimer's (5). Besides, many natural product biosynthetic enzymes are also members of this group (5). Structural characterization has been carried out for many such enzymes, among which are the rebeccamycin biosynthetic enzyme RebC (11), the prejadomycin hydroxylases PgaE and CabE (12), the aklavinone-11 hydroxylase RdmE (13), the prodeoxyviolacein hydroxylase VioD (PDB 3C4A), the anhydrotetracycline hydroxylase OxyS (14), the Diels-Alderase PyrE3 in the biosynthesis of pyrroindomycins (15), and the polyketide monooxygenase BexE involved in the BE-7585A biosynthesis (16). These structures have revealed that most enzymes possess dissimilar substrate-binding sites to adapt to their specific substrates, which have distinct properties in terms of chemical structure, size, shape, and polarity, and may utilize different strategies/ paths to recruit their respective substrates.
As a member of Group A flavin-dependent monooxygenases, PieE is responsible for the 4Ј-hydroxylation of the ␣-pyridone ring in the biosynthesis of piericidin A1 (Fig. 1a) (17). Owing to its structural similarity to coenzyme Q (ubiquinone), piericidin A1 is a potent inhibitor of NADH-ubiquinone oxidoreductase (complex I) of both eukaryotic and bacterial sources exerting broad antimicrobial and insecticidal activities (18,19).
In this work, we have structurally and biochemically characterized the unique hexameric monooxygenase PieE. Importantly, we have captured for the first time a novel "sliding" conformation of FAD upon substrate binding, in addition to the classical OUT and IN conformations. This novel snapshot of the mobile cofactor uncovers an important mechanistic step for Group A flavin-dependent monooxygenases and fills a gap currently missing in the orchestrated order of chemical events catalyzed by this important class of enzymes.

PieE forms a unique hexamer
In both PieE-FAD and PieE-FAD-substrate crystals, each asymmetric unit contains six subunits of PieE. The polypeptide chains of these 12 independent subunits are almost identical with an r.m.s.d. of 0.20 -0.35 Å for the 580 C␣ atoms, indicating no significant deviations among the subunits in the same crystal and also no drastic conformational change upon substrate binding. Each PieE subunit contains three domains: the FAD-binding domain (residues 7-82, 103-203, and 300 -374), the middle domain (residues 83-102, 204 -299, 375-432) for substrate binding, and the C-terminal domain (residues 433-586) (Fig. 1b).
Each asymmetric unit contains six subunits forming a hexamer, consistent with the results obtained through size-exclusion chromatography. Whereas highly similar tetrameric HbpA (41% identity) could be described as a dimer of dimers (21,24), the PieE hexamer is a trimer of dimers. Of note, the basic "dimer" unit in both enzymes has similar architecture, as shown in Fig. 1c. The two subunits in the dimer are organized through a "head-to-tail" style (i.e. the middle domain of the first subunit interacts with the C-terminal domain of the second subunit, and vice versa). In the dimerization interface, there are 12 H-bonds established, and the residues involved include Asn 85 , Ala 93 , Arg 99 , Arg 417 , Glu 481 , Arg 484 , Tyr 539 , and Glu 550 . The short helix consisting of Pro 107 -Ala 116 in the FAD-binding domain is located next to the pseudo-2-fold axis and also contributes to the formation of dimer via van der Waals contacts. In this dimerization interface, the buried surface area is around 1300 Å 2 . Three dimers are further assembled into a hexamer (Fig. 1c) such that, as shown in the side view, each dimer constitutes one side of the equilateral triangle and interacts with the other two dimers. Of note, all of the three domains of each subunit contribute to form the interface between dimers, which is stabilized by 14 H-bonds involving the residues Thr 47 , Gln 138 , Gln 144 , Arg 146 , Thr 170 , Arg 219 , Arg 401 , Asn 482 , and Gly 532 . The buried area between any two dimers is about 1500 Å 2 .
It should be pointed out that the PISA program failed to predict the hexameric form of PieE, although it could achieve 80 -90% accuracy for the prediction on the oligomeric state of macromolecular assemblies (25). Nevertheless, using different crystallization conditions, we have also obtained two other crystal forms of FAD-bound PieE that diffracted to ϳ3.4 Å (data not shown). The first one, albeit in the same P212121 space group as those reported here, has completely different unit cell parameters; the second crystal form is in the C2221 space group. Importantly, the same hexameric architecture of PieE has been observed in these two additional crystal forms.
To confirm quantitatively the hexameric form observed in the crystal structures, we performed size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis on PieE, enabling us to determine the absolute molecular mass of the homooligomer. MALS analysis of the single monodispersed peak that eluted from the size-exclusion chromatography column exhibited a molecular mass of 389

Sliding FAD in Group A flavin-dependent monooxygenases
kDa, corresponding to the molecular weight of a PieE hexamer ( Fig. S2 and Table S2). Small-angle X-ray scattering (SAXS) is another biophysical method capable of assessing the molecular weight and oligomeric state of biomolecules in solution (26). Using size-exclusion chromatography coupled to SAXS (SEC-SAXS), additional structural factors for PieE in solution were elucidated (Fig. 2a). Consistent with the crystal structure solved, a pair-distance distribution function calculated from the scattering of PieE in solution gave a radius of gyration of 47.3 Ϯ 0.1 Å and a maximum dimension of 132 Å ( Fig. 2 (b and c) and Table S3). From the SAXS data, Porod volume was derived to be 6.24 ϫ 10 5 Å 3 , which corresponds to an approximate molecular weight of 390 kDa (M r ϭ V p /1.6). Whereas the latter calculation is generally acceptable within a 25% margin, additional molecular weight estimation based on the SEC-SAXS data using the method of Fischer et al. (27,28) resulted in a molecular weight of 387 kDa (using q max ϭ 0.15 Å Ϫ1 ), in agreement with the mass of a hexamer, as observed by SEC-MALS.
Excluding the symmetry determined from the structure of PieE, an ab initio shape reconstruction using the DAMMIF algorithm of the ATSAS suite (29) (refined using DAMMIN) produced a dummy atom model fitting well the experimental data ( 2 ϭ 1.050, Fig. 2d, Fig. S3, and Table S3). Alignment of the refined dummy atom model with the crystal structure of PieE using SUPCOMB showed a good agreement in the shape and volume of the two structures (normalized spatial discrepancy ϭ 0.9379, Fig. 2d).
To the best of our knowledge, PieE is the first reported hexameric FAD-dependent monooxygenase. In fact, among 2194 FAD-bound structures in the Protein Data Bank, only one structure (PDB 2FG9) is annotated as a hexamer, but this putative antibiotic resistance protein (BT_3078) does not bear any similarity to PieE.

FAD adopts an "out" conformation in the PieE-FAD binary complex
The FAD molecule is bound to PieE in an elongated conformation with the ADP and ribityl moieties tightly anchored by the FAD-binding domain through many direct or water-mediated H-bonds (Fig. 3). Stabilization of the FAD isoalloxazine ring is mainly achieved by the side chain of Trp 296 (Fig. 3b), which stacks on its re-face through parallelinteractions, whereas its si-face is packed against Arg 52 and Ala 53 .
Inspection of the FAD-binding environment indicates that the cofactor molecule adopts an OUT conformation in the PieE-FAD complex (Fig. 3), similar to the substrate-free structures of HbpA (PDB 4Z2R) (21) and RebC (PDB 2R0C) (11), where the FAD isoalloxazine ring is also wedged between a tryptophan residue and an arginine residue (Trp 293 and Arg 46 in HbpA or Trp 276 and Arg 46 in RebC) (Fig. S4).

The Mer-A2026B substrate-binding pocket is preformed
Starting at the interface between the middle domain and the FAD-binding domain, there is a tunnel ϳ6 Å wide and ϳ18 Å long that extends to the protein surface (Fig. 4). The electron density maps of the PieE-FAD-substrate ternary complex, obtained through soaking the PieE-FAD crystals in the presence of Mer-A2026B, unambiguously confirmed the binding of substrate in this tunnel (Fig. 4). Consistent with the largely apo-

Sliding FAD in Group A flavin-dependent monooxygenases
lar nature of the substrate molecule, this cavity is lined by a significant number of hydrophobic residues, such as Leu 55 , Ala 206 , Leu 228 , Met 230 , Val 243 , Val 245 , Leu 256 , Phe 258 , Pro 323 , Met 378 , Leu 381 , and Ile 382 . As the substrate molecule is stabilized by a cluster of residues, removal of a single side chain may affect the optimal binding but is less likely to abolish the activity. This is substantiated by the fact that neither the L228A nor the F258A mutation completely inactivated the enzyme (Fig. 5). Nevertheless, introducing a bulky charged residue here, as evidenced by the activity measurement of V245R, has yielded an inactive enzyme (Fig. 5). Notably, in addition to the hydrophobic interactions to bind the long polyene tail, the 3Ј-hydroxyl group on the ␣-pyridone ring of substrate forms an H-bond with the imidazole ring of His 54 , and the hydroxyl group on the polyene tail is also within hydrogen-bonding distance of the main chain carbonyl of Ala 239 (Fig. 4). Notably, both the H54A and H54N mutants are inactive (see below) ( Fig. 5). As described below, the substrate molecule is located in the proximity of the FAD molecule, adopting the IN conformation.
In contrast to its structural homologue RebC, for which a remarkable reorganization of local structure (helix melting) is required to accommodate the substrate (11), the substratebinding pocket of PieE does not undergo large conformational change upon ligand binding despite the relatively large size of the substrate.

The signal of substrate binding is transmitted via subtle structural changes to the FAD-binding site
Despite the absence of large conformational change, the presence of substrate in the PieE tunnel has brought about noticeable differences in the local region, which has further led to a cascade of structural adjustments, as compared with the PieE-FAD binary complex (Fig. 6). First, the substrate-binding event has initiated a small but discernable movement of the ␤-strand harboring residues Leu 256 and Phe 258 , two residues shaping the substrate entry tunnel, with a displacement of 0.3-0.8 Å of polypeptide backbone. This signal has been passed on to the adjacent ␤-strand containing residues Ala 206 , Arg 207 , and Ala 208 , the C␣ atoms of which are displaced by 0.6 -0.8 Å. Subsequently, such changes are further propagated to the next ␤-strand, causing a shift of 0.8 -0.9 Å for the C␣ atoms of Trp 296 and Glu 297 . Finally, accompanying this main-chain  Table S3). b, buffer-subtracted averaged SEC-SAXS scattering profile of PieE with the Guinier region shown in the inset (estimated R g of 48.3 Ϯ 0.3 Å, Table S3). c, pair-distance distribution function (P(r)) from the SAXS profile of PieE exhibited an R g of 47.3 Ϯ 0.1 Å and a maximum dimension (D max ) of 132 Å. The back-calculated SAXS profile from the P(r) function is fit to the experimental data in the inset. d, the refined ab initio dummy atom model calculated from the pair-distance distribution function is overlaid with the hexameric structure of PieE.

Sliding FAD in Group A flavin-dependent monooxygenases
movement is a flipping of the Trp 296 side chain, which is now anchored through an H-bond by Thr 210 (Fig. 6) in the preceding ␤-strand and no longer located directly above the FAD molecule, resulting in the loss of parallel stacking to the isoalloxazine ring. The angle between the Trp side chain and the isoalloxazine ring in the ternary complex is estimated to be ϳ30°. Interestingly, the last two ␤-strands mentioned above are the starting and finishing ␤-strands that connect the middle domain to the FAD binding domain. As recently pointed out (30), the correlated backbone motions are a fundamental property of the ␤-sheet and are of pivotal importance to transfer signal of structural changes. Here, through a cascade of small but important conformational changes, the signal of the substrate-binding event has been delicately transmitted to the FAD binding site, triggering a flipping of Trp 296 and the loss of optimalinteractions with the isoalloxazine ring.

FAD adopts an IN conformation in five of six subunits in the PieE-FAD-substrate ternary complex
Comparison of the PieE binary and ternary complexes has revealed that, following the substrate-binding event and loss of the optimalinteractions with Trp 296 , the isoalloxazine ring of FAD has been observed to adopt the IN conformation in five of six subunits, namely chains A, B, D, E, and F, with the electron density maps for the isoalloxazine ring of FAD in chains A, B, and E being of good quality ( Fig. 7a and Fig. S5), whereas those for chains D and F are weaker, indicating a more mobile ring in these latter two chains. In general, the isoalloxazine ring of all of these FAD molecules has exhibited higher-temperature factors as compared with the ADP moiety or with those in the binary complex. To account for this, an occupancy of 0.8 is set for the IN conformation of the isoalloxazine ring, whereas the ADP moiety remains at full occupancy.

Sliding FAD in Group A flavin-dependent monooxygenases
The isoalloxazine ring in the IN conformation, stabilized by several H-bonds with surrounding residues, is suitable for the transfer of the -OH group from C4a-hydroperoxyflavin to the C4 position of the substrate ␣-pyridone ring, as evidenced by a relatively short distance of 4.5 Å between these two atoms. Above the isoalloxazine ring, there is constantly strong density (stronger than the ring and nearby water molecules) to which we have assigned a chloride ion (Fig. 7a), consistent with the abundance of chloride ion in both protein buffer (150 mM NaCl) and reservoir solution (200 mM MgCl 2 ). This chloride ion has direct contacts with the pyrimidine moiety of the isoalloxazine ring and is stabilized by the main chain amide groups of Leu 327 and Gly 328 as well as the side chain amide of Asn 321 , similar to the previously reported Group A members, such as PHBH (PDB 1DOC) (7), 3-hydroxybenzoate 6-hydroxylase (3HB6H) (PDB 4BJY) (31), and KMO (PDB 5NAK) (32), on which chloride has inhibitory effects. This is in stark contrast to the PieE-FAD binary complex, for which the electron density maps do not support the inclusion of a chloride ion at this position, despite the same amount of chloride ion present for both. As discussed below, the presence of chloride most likely coincides with the oxidized FAD molecule adopting an IN conformation.
It is notable that the FAD molecules in the IN conformation are best refined with a butterfly shape bent along the N5-N10 axis of the isoalloxazine ring, characteristics of a partially reduced state. This distortion from the planar structure is likely caused by its unique environment, which includes a chloride ion bound next to the isoalloxazine ring.

FAD is in a novel sliding conformation in chain C of the ternary complex
Unexpectedly, the isoalloxazine ring of FAD in chain C remained in the vicinity of Trp 296 despite the pronounced shift of the latter. To adapt to the change of Trp 296 upon substrate binding, the FAD molecule in this chain was observed to slide forward with a concomitant small tilting to maintain part of the interactions and van der Waals contacts with Trp 296 (Fig.  7b and Fig. S6). Upon superposition with the OUT conformation of FAD in the binary complex, a displacement of 0.9 -1.2 Å was observed for the atoms on the isoalloxazine ring, whereas the adenosine moiety only undergoes a slight movement of 0.3 Å owing to the flexibility of the ribityl chain. This new conformation brings the N3 atom of the isoalloxazine ring within hydrogen-bonding distance (moved from 4.0 to 3.5 Å) of the 3Ј-OH group of the substrate ␣-pyridone ring (Figs. 7b and 8a). Remarkably, different from the OUT conformation buried by the Trp side chain (Fig. 3b), the isoalloxazine ring is now more exposed to the solvent in this new position (Figs. 7b and 8 (a and  b)). Despite the modest displacement (0.9 -1.2 Å) of the isoalloxazine ring relative to that in the OUT conformation, small conformational changes often have profound consequences for protein function, as previously pointed out (33). To the best of our knowledge, this new sliding conformation attendant to the adjustment of an aromatic side chain above the FAD ring has not been previously reported. Notably, upon careful inspection of the potential effects of crystal packing on the flavin conformation, we did not find any specific crystal packing

Sliding FAD in Group A flavin-dependent monooxygenases
interactions that will restrict subunit C (sliding FAD) versus the other subunits (IN FAD), and, particularly, the binding environment for FAD does not seem to be affected by crystal packing. Therefore, the new conformational state observed here, namely sliding FAD together with the flipping of Trp 296 , should be biologically relevant and adopted by the enzyme in the catalytic cycle (Scheme 1). In this context, the structure of chain C has provided an impressive snapshot of how the FAD molecule chases after the shift of one neighboring residue (Trp 296 here), a domino effect initiated by the substrate binding.
Coincident with this sliding conformation of FAD in this chain C, the electron density corresponding to the chloride ion in the other five subunits is weaker here, and thus one water molecule was modeled here. It should be pointed out that, other than the difference in the FAD conformation and the presence or absence of chloride ion, no other major structural discrepancy was observed between the chain C and the other five chains. This strongly indicates that the sliding conformation is an intermediate state situated between the OUT conformation and the IN conformation (Fig. 8c). It is very possible that the sliding conformation and the IN conformation have a small difference in free energy. As previously suggested for PHBH in which the presence of bromide ion is linked to flavin orientation (7), the presence of chloride ion likely shifts the equilibrium between the conformational states of FAD toward the IN conformation, due to its interactions with the pyrimidine moi-  The chloride ion is shown as a magenta sphere. b, the FAD molecule adopts a unique sliding conformation in chain C of the ternary complex. In this binding mode, due to the change of orientation of Trp 296 , the interactions between Trp 296 and FAD are significantly weaker as compared with the binary complex, where the Trp 296 side chain and the isoalloxazine ring of FAD establish parallelstacking interactions (Fig. 3). The N3 atom of the isoalloxazine ring establishes a hydrogen bond with the substrate molecule. Hydrogen bonds are shown in dashed lines. The Fourier difference maps (F o Ϫ F c ) with ligands omitted from refinement are shown in green mesh and contoured at 2.5.

Sliding FAD in Group A flavin-dependent monooxygenases
ety of the IN FAD, as the latter is more populated in this hexamer crystallized at a high concentration of chloride.

Substrate binding stimulates NADPH oxidation
Previous studies on many other Group A monooxygenases, such as PHBH, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO), and Tet (50), have indicated that substrate binding could significantly promote the reduction of FAD by NAD(P)H (8,34,35,36). Our assays on PieE have shown similar tendency (Fig. 9). Whereas NADPH is oxidized very slowly in the buffer, the oxidation rate is increased with the addition of PieE. Expectedly, the rate of NADPH oxidation is 10 times faster in the presence of substrate than in its absence, confirming that substrate binding leads to a more solvent-accessible isoalloxazine ring facilitating the binding of nicotinamide ring of NADPH, the manner of which remains to be further investigated, and subsequent hydride transfer. On the other hand, the further addition of 200 mM NaCl eliminates the effects brought by the presence of substrate, which is consistent with our structural observation that the presence of chloride ion defers the switch of oxidized FAD from the IN conformation back to the OUT one for a new cycle of catalytic events and thus slows down the process. Importantly, the NADPH oxidation rate was remarkably affected by the indole side chain of Trp 296 . Removal of this side chain has resulted in significant decoupling of NADPH oxidation and substrate binding, leading to a less active hydroxylase (Fig. 5). This further demonstrates the pivotal role of Trp 296 in sensing substrate entry and triggering the FAD sliding required for efficient hydride transfer.

His 54 deprotonates substrate facilitating hydroxylation
Located at the bottom of the substrate binding tunnel is His 54 , whose imidazole ring forms an H-bond with the 3Ј-OH group of ␣-pyridone ring of the substrate (Fig. 4a). Its mainchain amide group also stabilizes the IN conformation of FAD through hydrogen bonding to the O4 atom of the latter. This arrangement has placed the 3Ј-OH group of the substrate ␣-pyridone ring at a distance of 3.6 Å from the N5 atom of FAD. Given the scarcity of polar atoms on the substrate molecule, His 54 is the only residue that forms hydrogen bonds to both substrate and cofactor, thus playing a bridging role in PieE catalysis through proper orientations of both partners in the catalytic site. Nevertheless, most likely, His 54 plays a more important role in substrate deprotonation. It has been suggested that, for many aromatic substrates in Group A monooxygenases, the deprotonation step is of critical importance, as it increases the nucleophilic property of the phenolic carbon to facilitate the ortho-hydroxylation of the substrate. Indeed, the H54N and H54A mutations have completely abolished the activity of PieE (Fig. 5), similar to the behavior of the H48A mutant in HbpA (24).
Following the substrate binding event and subsequent deprotonation by His 54 , the lack of proton on the 3Ј-O(H) moiety has pronounced effects: on one hand, the unprotonated substrate will likely await the binding of a reduced FAD in the IN conformation with a protonated N5 atom to form an H-bond; on the other hand, such a deprotonated state of the substrate will better accommodate the sliding conformation of

Sliding FAD in Group A flavin-dependent monooxygenases
oxidized FAD as described above, as the latter brings the N3 atom (with proton) close enough for a potential hydrogen bonding interaction. With this reasoning, the mechanism of how substrate binding could promote FAD reduction is clear. First, a cascade of small but pronounced conformational changes leads to the sliding conformation of FAD, which is

Scheme 1. Schematic representation of the putative order of binding and chemical events in the catalytic cycle of PieE and other pertinent Group A flavin-dependent monooxygenases.
The catalytic cycle is initiated by binding of the substrate, thus forming the enzyme-FAD-substrate complex. This event leads to the sliding conformation of FAD, followed by NADPH binding. Although it remains to be experimentally observed, upon NADPH binding, the oxidized FAD molecule most likely stays in the sliding conformation or in a very similar conformation (denoted as [SLIDING]). In this [sliding] conformation, oxidized FAD is reduced by NADPH, followed by the release of NADP ϩ . These steps constitute the reductive half-reaction. In the subsequent oxidative half-reaction, the reduced FAD shifts to the IN conformation, followed by the reaction with molecular oxygen to form C4a-hydroperoxyflavin. The next step is the monooxygenation of substrate to yield the hydroxylated product along with the formation of C4a-hydroxyflavin. A dehydration reaction leads to the release of the final product and water. This last step terminates the cycle with regeneration of the oxidized FAD returning to the OUT conformation. Importantly, the mobile flavin alternates in at least three different conformations, namely OUT, sliding, and IN, to fulfill the catalytic cycle.

Sliding FAD in Group A flavin-dependent monooxygenases
more approachable for NADPH binding. Second, deprotonation of substrate also favors the binding of reduced FAD in IN conformation as well as the placement of oxidized FAD in the sliding conformation. The regulatory role of substrate deprotonation is also important in PHBH, but the mechanism is different. In PHBH, the ionized substrate (phenolate) repels the main-chain carbonyl of Pro 293 , which triggers the movement of FAD from IN to OUT conformation for reductive half-reaction (37).

Discussion
We report here the crystal structure of PieE, the monooxygenase responsible for the hydroxylation step in the biosynthesis of piericidin A1. Most importantly, current structures have allowed experimental observation of three conformational states of FAD. In addition to the well-documented OUT and IN conformations, we have observed, for the first time, a sliding conformation of FAD upon substrate binding. This result uncovers an important mechanistic step in Group A enzymes and provides molecular insights into the mechanism of how the substrate-binding event facilitates NADPH binding and FAD reduction.
PieE represents a unique hexameric flavoenzyme. Notably, the C-terminal domain of PieE is heavily involved in forming the interface for dimers and for trimer of dimers. This C-terminal domain is present in a significant portion of Group A flavindependent monooxygenases. For some structural homologues of PieE, including HbpA (21), phenol hydroxylase (PHHY) (38), and 3-hydroxybenzoate hydroxylase (MHBH) (39), the C-terminal domain also contributes to the oligomerization. In contrast, for some other Group A members, such as CabE (12), OxyS (14), and RIFMO (22), their C-terminal domain is remote from the dimerization interface. Moreover, some Group A members (e.g. RebC (11) and RdmE (13)) are monomeric, although they have a similar C-terminal domain. Notably, oligomerization could be still achieved for some Group A members, such as DHPH (40) and MHPCO (41), that lack such a C-terminal domain. This indicates that the C-terminal domain is required neither for oligomerization nor for monooxygenation. As suggested previously (22), this domain is far from the FAD and substrate-binding sites and may play a role in protein folding and stability of these proteins. Further investigation is required to better understand this issue.
Since the seminal discovery of mobile flavin in PHBH more than 2 decades ago (7), dozens of Group A monooxygenases have been structurally characterized. Nevertheless, observations of both conformations for a single protein are still uncommon. So far, only a handful of proteins, such as PHBH (7), PHHY (38), RebC (11), HbpA (21,24), Tet(50) (42), and TropB (43), have both IN and OUT conformations (Table S1). Worthy of mention is that, for PHBH, both conformational states of FAD were modeled into one single subunit (PDB 1DOE), and the partial occupancy of FAD in IN conformation was attributed to the presence of bromide above the isoalloxazine ring, analogous to the chloride-binding site in PieE reported here. At the IN position, the FAD molecule was observed to undergo a "tilting" in response to the binding of a bulky inhibitor, as demonstrated by the recent crystal structure of the KMO (PDB 5NAG) despite the fact that the flavin is still in the canonical IN position when L-kynurenine, the bona fide substrate, is bound (PDB 5NAK) (32). Notably, an alternative conformation of FAD named the "open conformation," in the substrate-free R220Q mutant structure of PHBH (PDB 1K0L) was previously suggested to enable substrate entry to the active site (44). Up to now, such a conformation of FAD proposed for substrate entry in PHBH has not been observed in PieE and other monooxygenases. The open conformation of FAD in PHBH is therefore less likely to be a general mechanism, as distinct substrate binding/ entry paths have been observed for many other enzymes in this family (45).
In contrast to the Group B flavin-dependent monooxygenases, which have a dedicated domain to keep NAD(P)(H) bound during the catalytic cycle, Group A enzymes bind the reduced nicotinamide coenzyme less tightly, and NAD(P) ϩ is released immediately upon flavin reduction (46). This transient binding is likely the major reason why it is hard to trap NAD(P)(H) in the crystal structures of Group A members. To the best of our knowledge, the only NADPH-bound structure is that of the PHBH R220Q mutant (PDB 1K0J) (44). However, in this structure, NADPH binds in an extended conformation, and the C4 atom of the nicotinamide ring is 19.3 Å away from the N5 atom of the FAD isoalloxazine ring, which makes this conformation unfit for flavin reduction to occur. It was suggested but not confirmed that NADPH would adopt a folded conformation to bring its nicotinamide moiety closer to the isoalloxazine moiety of FAD to facilitate hydride transfer (44). Therefore, the productive binding mode of NAD(P)H in the Group A enzymes remains to be identified.
The sliding conformation reported here constitutes a critical addition to the repertoire of dynamic snapshots of FAD conformations and could be treated as an intermediate/transition conformation after substrate entry and before NADPH binding (Scheme 1). The space vacated by the flipping of Trp 296 side chain upon substrate binding will better accommodate NADPH in an orientation suitable for hydride transfer to FAD. It should be pointed out that, in the well-studied PHBH enzyme, there is no such aromatic residue analogous to Trp 296 in PieE, and therefore the FAD molecule in the OUT conformation is much more solvent-accessible. Despite the fact that 35 structures are available in the PDB for this enzyme, not a single substrate-free structure of WT PHBH is available; thus, the potential structural changes caused by substrate binding remain unclear. In a stark contrast, in PieE and many other Group A monooxygenases, the isoalloxazine ring of FAD in OUT conformation is snugly covered from the top by an aromatic residue Trp or Tyr (Fig. 3b and Fig. S4), as shown in the structures of PhzS (PDB 2RGJ) (47), RebC (PDB 2R0C) (11), HbpA (PDB 5BRT) (21), RdmE (PDB 3IHG) (13), Tet(50) (PDB 5TUE) (42), and MHBH (PDB 2DKH) (39). As pointed out in the previous reports on MHBH (39) and RebC (11), significant conformational changes of these aromatic side chains are required for NAD(P)H binding and subsequent FAD reduction. Notably, when Trp 276 in RebC adopts a conformation analogous to that of Trp 296 in the chain C of PieE, the FAD molecule in RebC is already in the IN conformation, leaving unanswered how FAD could await NADPH binding. The sliding conforma-

Sliding FAD in Group A flavin-dependent monooxygenases
tion reported here fills this gap for a significant number of Group A monooxygenases with different substrate profiles, providing a hint on how NADPH binding could be efficiently initiated through subtle structural changes induced by substrate entry in these enzymes. On the other hand, the presence of an aromatic residue on top of the isoalloxazine ring is likely beneficial for the overall cycle of chemical events leading to the monooxygenation of substrate. If the FAD molecule were more exposed, access of NADPH would be easier, and thus hydride transfer would be more frequent. This would lead to undesirable FAD reduction in the absence of substrate. Therefore, the conservation of an aromatic residue (Trp or Tyr) here is likely a protective mechanism to avoid wasteful NADPH oxidation, which is critical for efficient catalysis. Indeed, as recently demonstrated in the case of MHPCO (41), both Y270F and Y270A mutants showed elevated basal activity (NADH oxidation) compared with the WT enzyme, and the Tyr residue is required to increase the coupling of the NADH oxidation with ring cleavage.
As described previously, the majority of FAD molecules in the PieE-FAD-substrate ternary complex adopt the IN conformation upon substrate binding. This has coincided with the presence of a chloride ion above the pyrimidine moiety of FAD, consistent with the observation made with PHBH (7). Motivated by this linkage, we have carefully inspected a significant number (ϳ30) of pertinent proteins in PDB. For six proteins (PHBH (PDB 1DOE) (7), 3HB6H (PDB 4BJY) (31), KMO (PDB 5NAK) (32), PgaE (PDB 4ICY) (48), HpxO (PDB 3RP8) (49), and TropB (PDB 6NET) (43)), a chloride or bromide has been modeled above the FAD molecule. In fact, our analysis indicated that a chloride ion should also have been modeled above the isoalloxazine ring for many other structures of Group A monooxygenases, such as HbpA (PDB 4CY8), PHHY (PDB 1PN0), and PgaE (PDB 2QA1), to more reasonably account for significantly stronger electron density and lower temperature factors (when modeled as water) relative to the surrounding atoms. Importantly, all of these strong electron density peaks occur only when FAD adopts the IN conformation. As a matter of fact, FAD has been observed exclusively in the IN conformation for 17 proteins, whereas six other proteins have solely the OUT conformation, and another six have both conformations (Table S1). That is to say, the majority of proteins contain FAD in the IN conformation regardless of the substrate being present or not. Given that oxidized FAD was used for almost all of these structures (except one structure of RebC with reduced FAD (50)), a reasonable scenario is that oxidized FAD should move to the OUT conformation in the presence of substrate to initiate a new cycle of catalysis. We speculate that the abundant presence of the IN conformation is correlated to the frequent use of chloride in both protein buffer and reservoir solution, as exemplified by our current study, where the protein buffer and the reservoir solution contain 150 mM NaCl and 200 mM MgCl 2 , respectively. Probably, in some of the substrate-bound structures, oxidized FAD is stalled in the IN conformation due to chloride ion. Particularly, considering that the chloride ion fills the space predicted to hold the oxygen atoms of the flavin hydroperoxide intermediate produced from the reaction between the reduced flavin and molecular oxygen, it is very possible that this chloride ion will impede the binding and/or reactivity of O 2 within the enzyme active site as suggested previously (31,32). Therefore, it is plausible that structural characterization of this class of enzymes may benefit from utilization of protein buffers that do not contain a high concentration of chloride. Moreover, crystals obtained in the absence of chloride/bromide may be worthy of special attention. This may help elucidate the subtle mechanisms of these enzymes and increase our chances to visualize novel snapshots of the mechanistic steps in catalysis.
With the new sliding conformation observed here, we could better envision the concerted conformational changes of both protein and flavin required for NAD(P)H binding. To have an ultimate understanding of how the pyridine nucleotide coenzyme (NAD(P)H) is recognized, more efforts are required to tackle this unresolved issue for the Group A flavin-dependent monooxygenases.

Cloning, protein expression, and purification
The pieE gene from Streptomyces sp. SCSIO 03032 was cloned into the pET28a vector as described previously (17) to generate an N-terminal His tag fusion protein with a thrombin cleavage site. The expression and purification was done following a similar protocol used for CrmK (51). To remove the His tag by thrombin cleavage, PieE was buffer-exchanged to 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2% (v/v) glycerol, followed by an overnight incubation with thrombin (MP Biomedicals) at a molecular ratio of 1:100. The purified PieE fractions were pulled and concentrated for crystallization trials and enzymatic assays.

Site-directed mutagenesis of PieE
Site-directed mutagenesis of PieE was carried out using Agilent's mutagenesis kit according to the manufacturer's instructions. Six mutations, including H54N, H54A, L228A, V245R, F258A, and W296A, were generated and confirmed by Sanger sequencing. The plasmids carrying the mutated genes were transformed into Escherichia coli BL21 (DE3) for overexpression. The PieE mutants were purified using the same protocol for the WT PieE.

Crystallization, data collection, and structure determination
PieE at a concentration of 14 mg/ml was crystallized through the microbatch method in the reservoir solution containing 30% PEG 400, 0.2 M MgCl 2 , and 0.1 M HEPES, pH 7.5. The crystal of the PieE-substrate-FAD complex was obtained by soaking the PieE crystals in the reservoir solution containing 1 mM substrate. For data collection, the crystals were flash-cooled in the N 2 cold stream (Oxford Cryosystem, Oxford, UK).
Data for the PieE-FAD and PieE-FAD-substrate complexes were collected to 2.02 and 2.52 Å, respectively, at a wavelength of 0.9795 Å at the CMCF beamline, Canadian Light Source. The unit cell parameters are shown in Table 1. Data processing and scaling were performed with iMosflm (52). The structure solution was obtained by molecular replacement through MolRep (53) using the structure of HbpA (PDB 4Z2U, which shares 41%

Sliding FAD in Group A flavin-dependent monooxygenases
sequence identity with PieE) (21) as the search model. Several cycles of refinement using REFMAC5 (54) followed by model rebuilding with Coot (55) were carried out. For the substratebound PieE structure, the substrate molecule was placed in the model based on the Fourier difference map and refined using the geometric restraints prepared using REEL in Phenix (56). Both models have good stereochemistry as analyzed with PRO-CHECK (57). Data collection and refinement statistics for both structures are shown in Table 1.

Light and X-ray scattering of PieE
The molecular mass and the hydrodynamic radius of PieE were investigated by SEC-MALS using an ÄKTAmicro (GE Healthcare) FPLC system coupled in series to a Dawn HELEOS II multi-angle light scattering detector and an OptiLab T-rEX refractive index detector (Wyatt Technology). Simultaneous scattering from 18 detectors was recorded, and data were further analyzed using ASTRA 6.1.5.6 (Wyatt Technology). More data collection details are given in Table S2.
The radius of gyration (R g ), forward scattering (I(0)), Porod volume (V p ), and maximum dimension of PieE (D max ) were derived from SEC-SAXS data collected with an ÄKTAmicro (GE Healthcare) FPLC coupled to a BioXolver SAXS system (Xenocs) equipped with a MetalJet D2 ϩ 70 kV X-ray source (Excillum) and a PILATUS3 R 300K detector (Dectris). X-ray scattering images corresponding to 30 s of exposure were collected during the elution, and an average scattering profile of all frames within the elution peak was calculated. Buffer scattering was then subtracted from the elution peak average scattering profile to yield the scattering curve of the sample. More data collection details are given in Table S3.

Activity assays of PieE WT and mutants
Assays were conducted in a 50-l reaction mixture in 50 mM Tris-HCl, pH 8.0 containing 100 M substrate, 5 M enzyme (WT or mutant), 2 mM NADPH incubated at 25°C for 2 h. 50 M FAD was added in the mixture for the assay of the W296A mutant. Control assays contained no enzyme, and reactions were quenched by the addition of 50 l of MeOH, and denatured proteins were removed by centrifugation. The assays were monitored by HPLC analysis carried out on a reversedphase column ZORBAX C18 (Agilent, 150 ϫ 4.6 mm, 5 m) with UV detection at 230 nm under the following program: solvent system (solvent A, 0.05% TFA in water; solvent B, 100% CH 3 CN, 0.05% TFA); 10% B to 90% B (0 -23 min), 90% B maintain (23-25 min), 90% B to 10% B (25-26 min), 10% B (26 -27 min); flow rate at 1 ml min Ϫ1 .

NADPH oxidation assay of PieE
To investigate the oxidation of NADPH by PieE, we have carried out assays in the absence and presence of substrate (50 M) and additional chloride ion (200 mM NaCl). All reactions were carried out in 50 mM potassium phosphate, pH 6.5, and 100 M FAD. The enzyme concentration (mutant or WT) was set at 5 M. Reactions were initiated by the addition of 100 M NAPDH, and its oxidation was monitored at 340 nm.

Data availability
The coordinates and structure factors of PieE-FAD binary complex and PieE-FAD-Mer-A2026B ternary complex have been deposited in the Protein Data Bank with the accession codes 6U0P and 6U0S, respectively. The SAXS data reported in this paper have been deposited in the Small Angle Scattering