Structural definition of the lysine swing in Arabidopsis PNAS PLUS thaliana PDX1: Intermediate channeling facilitating vitamin B6 biosynthesis Graham C. Robinsona, Markus Kaufmanna, Céline Rouxa, and Teresa B. Fitzpatricka,1

aDepartment of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerland

Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved August 5, 2016 (received for review May 20, 2016)

Vitamin B6 is indispensible for all organisms, notably as the coen- zation, and aromatization to produce the PLP molecule has received zyme form pyridoxal 5′-phosphate. Plants make the compound de considerable attention but remains enigmatic (7–10). Both bio- novo using a relatively simple pathway comprising pyridoxine syn- chemical and structural studies have served to capture snapshots of thase (PDX1) and pyridoxine glutaminase (PDX2). PDX1 is remarkable thePDX1enzymeinactionatcertain stages along the catalytic given its multifaceted synthetic ability to carry out isomerization, trajectory (10–18). Nonetheless, the sequence of events cannot yet imine formation, ammonia addition, aldol-type condensation, cycli- be stitched together and is essential to provide a complete overview zation, and aromatization, all in the absence of coenzymes or recruit- of one of nature’s most complicated from a fundamental ment of specialized domains. Two active sites (P1 and P2) facilitate perspective, as well as its potential as a drug target. the plethora of reactions, but it is not known how the two are co- Multifunctional enzymes generally recruit distinct domains for ordinated and, moreover, if intermediates are tunneled between their reactions, or connect different sites by substrate channeling active sites. Here we present X-ray structures of PDX1.3 from Arabi- (19, 20), or remodel a single (21) to facilitate the dopsis thaliana, the overall architecture of which is a dodecamer of multitude of transformations taking place. Substrate channeling β α ( / )8 barrels, similar to the majority of its homologs. An apoenzyme can be mediated by tunneling (22) or by transfer via a covalently structure revealed that features around the P1 active site in PDX1.3 bound prosthetic group to the successive active site. Classic ex-

have adopted inward conformations consistent with a catalytically amples of the latter are lipoyl-lysine residues of 2-oxo acid dehy- BIOCHEMISTRY primed state and delineated a substrate accessible cavity above this drogenases (23), biotinyl-lysine residues of carboxylases (24), and active site, not noted in other reported structures. Comparison with phosphopantetheinyl-serine residues of fatty acid synthases (25, thestructureofPDX1.3withanintermediatealongthecatalytic 26). Notably, the referred to prosthetic groups are all vitamin- trajectory demonstrated that a lysine residue swings from the dis- derived coenzymes. The covalently attached prosthetic groups tinct P2 site to the P1 site at this stage of catalysis and is held in place generally have freedom to rotate within their respective active by a molecular catch and pin, positioning it for transfer of serviced sites and thus serve as swinging arms ferrying substrates or inter- substrate back to P2. The study shows that a simple lysine swinging mediates to the subsequent active site (27). Enhancement of arm coordinates use of chemically disparate sites, dispensing with catalytic efficiency, channeling, and protection of stable interme- the need for additional factors, and provides an elegant example diates are hypothesized reasons for such approaches (28). None- of solving complex chemistry to generate an essential metabolite. theless, the concept has remained open to questions, which include whether the decorated side chain swings or the whole Arabidopsis thaliana | vitamin B6 biosynthesis | crystal structure | protein domain, as well as the need for the posttranslational lysine swing Significance itamin B6 in its form as pyridoxal 5′-phosphate (PLP) is an Vessential coenzyme involved in over 140 biochemical reactions, more than any other known nutrient. It is the most versatile coen- Multifunctional enzymes have been shown to recruit distinct do- zyme known in nature being involved in transamination, de- mains for their reactions, remodel active sites, or connect different carboxylation, elimination, racemization, and replacement reactions sites by substrate channeling to facilitate the multitude of trans- (1). It is biosynthesized de novo by microorganisms and plants. As an formations taking place. Pyridoxine synthase (PDX1) of the vitamin essential micronutrient, it must be taken in the diet of animals in- B6 biosynthesis machinery is a remarkable that alone has cluding humans. The absence of the pathway in animals renders the a polymorphic catalytic ability designated to two active sites, the biosynthesis de novo pathway a potential drug target in pathogenic coordination of which is unclear. Here structural snapshots allow organisms (2). It is now established that there are two biochemical us to describe a lysine swinging arm mechanism that facilitates routes that lead to the biosynthesis de novo of PLP. The first to be serviced substrate transfer and demonstrates how an enzyme can resolved was the deoxyxylulose 5-phosphate (DXP)-dependent path- couple distinct chemistry between active sites, dispensing with way, which requires seven enzymes and is found in a few select the need for extra domains, substrate tunneling, or transfer of microorganisms including Escherichia coli (reviewed in ref. 3). The coenzyme bound intermediates. The work provides an elegant ’ second route does not involve DXP (DXP-independent), only re- example of simplicity at work in nature s sea of complexity. quires two enzymes, and is by far the predominant route being found Author contributions: G.C.R. and T.B.F. designed research; G.C.R. and M.K. performed re- in most microorganisms and all plants (reviewed in ref. 3). The two search; G.C.R., M.K., and C.R. contributed new reagents/analytic tools; G.C.R. and T.B.F. enzymes required for the DXP-independent route are pyridoxine analyzed data; and G.C.R. and T.B.F. wrote the paper. synthase (PDX1) and pyridoxine glutaminase (PDX2), which to- The authors declare no conflict of interest. gether form a glutamine amidotransferase complex that uses ribose This article is a PNAS Direct Submission. 5-phosphate (R5P), glyceraldehyde 3-phosphate (G3P), and gluta- Data deposition: Crystallography, atomic coordinates, and structure factors have been mine in a highly complicated sequence of reactions to produce PLP deposited in the RCSB Protein Data Bank [accession nos. 5K3V (apo-PDX1.3) and 5K2Z (4, 5) (Fig. 1). Although PDX2 is a classic glutaminase hydrolyzing (PDX1.3-adduct)]. glutamine to ammonia and glutamate (4–6), the remarkable reaction 1To whom correspondence should be addressed. Email: [email protected]. mechanism of PDX1 that involves pentose and triose isomerization, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. imine formation, ammonia addition, aldol-type condensation, cycli- 1073/pnas.1608125113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1608125113 PNAS Early Edition | 1of9 Downloaded by guest on October 1, 2021 OH OH NH catalysis. A molecular catch and pin mechanism explains the re- H2N O O 2 O 2- OPO3 straint of this lysine in P1, release of which presumably facilitates HN OH OPO 2- the swing back to P2 and completion of PLP biosynthesis. Overall, R5P OH 3 K97 NH HN these snapshots of the plant PDX1 provide key insights into the O K165 K165 G3P intricate workings of a remarkable enzyme essential for plant P + H O 2H O survival and more generally into multifunctional enzymes. NH3 i 2 2 H2O N OH Results and Discussion

2- O OH OPO3 Arabidopsis 2- PDX1.3 from Adopts a Dodecameric Architecture. The

OPO3 O structure of apo-PDX1.3 from Arabidopsis has been determined at OH NH PLP NH 2 1.9 Å resolution (R ,17.7%;R , 20.3%; Table S1). It com- P1 P2 work free K97 K165 prises residues 20–296 with 13 residues not visible at the C ter- minus in addition to the 19 missing from the N terminus. Twelve Fig. 1. Scheme depicting selected reactions of PDX1 in the P1 and P2 active β α sites. Ribose 5-phosphate (R5P) enters the P1 active site of PDX1 and forms a PDX1.3 subunits that fold as ( / )8 barrels form a double hex- covalent intermediate with K97 (Arabidopsis numbering). Loss of water and americ ring interdigitating into the dodecameric structure

inorganic phosphate (Pi) from R5P and incorporation of ammonia (NH3) (Fig. 2A). The dodecameric architecture is consistent with earlier leads to formation of a covalent chromophoric adduct. The presence of K165 estimations of the molecular mass of PDX1.3 (411,000 Da) by size in the P1 site at this stage of catalysis facilitates transfer of the chromophoric exclusion chromatography coupled to static light scattering (30). adduct to the P2 active site. Condensation with glyceraldehyde 3-phosphate The individual hexameric rings are 100 Å in diameter with a (G3P) to form the product pyridoxal 5′-phosphate (PLP) completes one cat- central pore 40 Å in diameter (Fig. 2A). The two hexamers stack e alytic cycle. The interactions of the -amino groups of the respective lysines together to form a structure 90 Å high (Fig. 2A). The overall ar- with either R5P or the derived chromophoric adduct are depicted in gray. chitecture is similar to homologs of PDX1.3 that have been crys- tallized from bacterial sources including Bacillus subtilis (2NV1 modification to include the prosthetic group facilitating the and 2NV2) (16), Geobacillus stearothermophilus (1ZNN, 4WXY, multistep chemical reaction (28). The complex chemistry carried 4WXZ, and 4WY0) (15, 29), Thermotoga maritima (2ISS) (17), out by PDX1 provides an intriguing example to investigate in and Mycobacterium tuberculosis Rv2606c (4JDY) (31), as well as the context of multifunctional enzymes given that no prosthetic those from apicomplexan Plasmodium species (4ADS, 4ADT, and group is required and the enzyme alone carries out the entire 4ADU) (11), all of which form dodecamers. Notably, structures of series of reactions. the homologous enzymes from the yeast Saccharomyces cerevisiae All PDX1s are made up of two active sites referred to as P1 and (3FEM, 3O05, 3O06, and 3O07) (13, 18) and the archaeon Pyrococcus horikoshii (4FIQ and 4FIR) (32) crystallize as hex- P2 (16, 17, 29) and together coordinate the sequence of reactions α β resulting in PLP production (Fig. 1). The process begins with the amers. Labeling of individual -helices and -strands of PDX1.3 binding of R5P in the P1 site in a covalent interaction with an es- was done according to the labeling introduced for the B. subtilis sential lysine residue [K97 in Arabidopsis thaliana (hereafter referred homolog (Fig. 2B) (16). Thus, contact between each adjacent (β/α) barrel in PDX1.3 is established by helix α8′′, which runs to as Arabidopsis) PDX1.3] (10, 17). Loss of phosphate and water 8 parallel to helix α8 near the C terminus (Fig. 2 A and B), as for from R5P as well as the incorporation of ammonia (from glutamine all homologs. As for other dodecameric PDX1s, the interdigi- in the presence of PDX2) result in a chromophoric intermediate tation of the hexameric rings in PDX1.3 is established by helices likely to be unique to PLP synthase, which has been observed α6, α6′,andα6′′ of each subunit (Fig. 2 A and B). Notably, helix spectroscopically (8–10) but has eluded precise structural charac- αN is present despite the absence of the glutaminase subunit. terization. The reaction can only proceed further in the presence of Previously, this helix has only been observed in complexes of G3P, which is assumed to take placeintheP2siteandiswherethe PDX1 with PDX2, with the exception of PDX1 from S. cer- final product PLP has been observed in X-ray crystal structures (10, evisiae (13, 16, 17). 12, 18). A pertinent question is how the intermediate is transferred from the P1 to the P2 site. A second lysine (K165 in Arabidopsis The P1 Active Site of apo-PDX1.3 Displays a Catalytically Poised PDX1.3)ispostulatedtoorchestrate the interaction between the Architecture. Two active sites, annotated P1 and P2, have been active sites (10, 17) but had only been observed pointing toward the definitively located in PDX1 enzymes (16, 17). These sites were P2 site until recently. The recent report captured PDX1 in a rather originally defined by bound chloride, phosphate, or sulfate ions in heterogeneous population of different catalytic states in which this X-ray crystallographic structures (16, 17, 29) and more recently by second lysine points into either theP1orP2site(15);however,the the bound substrate R5P (P1) and product PLP (P2) (11, 18, 32) mixture of states in each subunit precluded clarification of the and have been validated through several biochemical studies (10, transition between the two states. Precise definition of the swinging 12, 16). In PDX1.3, the P1 site is located at the C-terminal mouth of this essential lysine between the active sites would help to resolve of the β-barrel and contains density for a sulfate ion surrounded by the obscure steps between the initiation of the PLP synthase reac- the characteristic phosphate-binding loop that includes Gly–Thr– tion in P1 and its completion in P2. Gly, as well as other established conserved residues of this active Here we have determined X-ray crystal structures of a PDX1 site (16, 29) (Fig. 2B). The P2 site is located at the interface be- (PDX1.3) from Arabidopsis. Like its bacterial counterparts, the tween subunits of opposing hexamers and also contains density for plant protein displays dodecameric architecture. In contrast to a bound sulfate ion surrounded by the conserved residues char- previous structures, the Arabidopsis apoenzyme is in a closed acteristic of this region (Fig. 2B). As for the homologous coun- conformation but poised for catalysis and serves to delineate a terparts of PDX1.3, the bound sulfates in P1 and P2 occupy the cavity above the P1 active site that provides access to the R5P same position as the phosphates of bound R5P and PLP, re- substrate. In the apoenzyme, the second active site lysine is ori- spectively (11, 15, 17, 18, 32). Markedly, except for coordinated ented toward the P2 site defining the resting state, i.e., its position waters, there is no other density observed in the P1 or P2 sites, at the beginning and end of the catalytic cycle. The structure of such that this structural snapshot of the PDX1.3 enzyme can be Arabidopsis PDX1.3 with the chromophoric intermediate solved to considered substrate and product free, i.e., apo-PDX1.3. 1.8 Å was also determined and can be used to corroborate a Although the overall architecture of PDX1.3 from Arabidopsis proposed structure for this adduct. Moreover, this structure clearly is similar to that of its homologs (rmsd of 1.14 Å, 0.77 Å, and placed the second active site lysine in the P1 site at this stage of 1.35 Å comparing 250, 262, and 242 corresponding Cα atoms of

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1608125113 Robinson et al. Downloaded by guest on October 1, 2021 A located at the N terminus of this helix in PDX1.3, S252, forms a PNAS PLUS hydrogen bond with the amide of A66, located at the N terminus of α2′, as well as interacting with the dipole moment of α2′ (Fig. 3C). In contrast to the other reported PDX1 structures, we also 40 Å 90 Å observed that most of the C-terminal region (residues 286–296 out 90º of 308) was resolved in PDX1.3 (Fig. 3A), even though the sub- strate is absent. The C-terminal region following on from α8′′ extends from the outer surface of the subunit in the dodecamer to the lumen across a cleft formed between adjacent subunits in the 100 Å same hexamer (Fig. 3A). Remarkably, the peptide backbone of β1 P-loop this region is zippered into place by intrasubunit and intersubunit B β2 G169 G171 interactions across this cleft (Fig. 3D). On one side, a series of α2 α1 hydrogen bonds comprising the guanidinium group of R263 and β3 D40 α8 M59 K97 amide groups of G253, G258, and T170 within the same subunit P1 site T170 α2' α8' β4 coordinate the peptide backbone of the C-terminal region directly β3 α3 D118 F249 β8 β2 β1 α8'' or through coordination of waters. This is matched on the other β8 M161 β4 αN β5 β7 side by interaction with the side chains of R76 and D79 and the β5 β6 amide of V74 located in the loop connecting α2′ and α2ofthe β6β7 α7 α4 H131 β6 adjacent subunit, in addition to a network of structural waters (Fig. 3D). α5 α6 K165 R154 Taken together, the inward position of α2′ as well as α8′ and the P2 site α6'' coordination of the C-terminal region define three important α5 structural arrangements that have only been collectively seen α6' α6' R153 K203 previously in PDX1 structures in the presence of the R5P sub- strate. As these arrangements serve to limit access to the P1 site, they are assumed to represent a catalytically operating state. BIOCHEMISTRY Fig. 2. Overall architecture of PDX1.3 from Arabidopsis.(A) PDX1.3 forms a However, we have observed this arrangement in the absence of dodecamer composed of two interdigitated hexamers (green and light blue), substrate and must therefore conclude that this rather represents a shown from above and the side after rotation by 90°. Dimensions are as in- catalytically poised state, at least in the case of PDX1.3. Moreover,

dicated. (B) The individual subunits of PDX1.3 adopt a (β/α)8 fold with addi- the network of intrasubunit and intersubunit interactions zipper- tional structural elements labeled as indicated. The P1 and P2 catalytic sites can ing the C-terminal region in place would appear to be poised to be clearly defined with residues characteristic of each site and sulfate ions coordinate cross-talk between two neighboring subunits. We have (yellow and red sticks) shown. Note one residue in P2, lysine 203 (K203), is previously demonstrated that PDX1 displays high in contributed by α6′ of a subunit on the opposing hexamer (light blue). Waters σ relation to the binding of the R5P substrate for which the C ter- have been omitted for clarity. The electron density is contoured to 1.0 . minus is essential (12, 14). Therefore, this structure of PDX1.3 serves to capture the fundamental essence of the C terminus in B. subtilis, G. stearothermophilus,andS. cerevisiae PDX1, respectively), linking neighboring catalytically poised subunits. we observed that the small segment of each subunit inserted A Catalytically Operational PDX1.3 Captures a Lysine Swinging Arm between β1 and α2 within the lumen of each hexameric ring, Mechanism. To further test if the geometric arrangements observed comprising residues 65–71, adopts an ordered conformation, α2′, in PDX1.3 represent a catalytically poised state rather than a state in PDX1.3 from Arabidopsis (Fig. 3A). In the reported bacterial α ′ closed to substrate (possibly caused by bound sulfate mimicking PDX1 structures, the 2 fold has only been observed in struc- substrate binding), we performed crystal-soaking experiments with tures of the ternary complex with the glutaminase PDX2 and R5P. Indeed, it must be mentioned that the P1 site is predomi- was, moreover, suggested to result from priming by a signal from – α ′ nantly hydrophobic, the majority of residue interactions only being the glutaminase subunit (15 17). However, 2 has since been with the phosphate moiety of the substrate. Because an am- observed in the reported structure of PDX1 alone from the monium salt is present in the crystallization solution, we an- archaeon P. horikoshii (4FIQ and 4FIR) (32), as well as in the ticipated that the enzyme would catalytically process R5P eukaryotic PDX1 structures reported from S. cerevisiae (3FEM, under these conditions. Specifically, we have shown previously 3O05, 3O06, and 3O07) (13, 18) and Plasmodium berghei (4ADT that PDX1 can transform covalently bound R5P into a chro- and 4ADU) (11), all of which were crystallized in the absence of mophoric intermediate with an absorption maximum around PDX2. Furthermore, in the archaeal and eukaryotic structures, 315 nm that results from loss of water and phosphate in the α ′ 2 exists in either of two conformations (out or in), dependent presence of a source of ammonia (10) (Fig. 1). We verified that on the absence or presence of R5P, respectively (11, 18, 32). In this intermediate forms under the crystallization conditions the presence of the substrate, the inward movement of α2′ par- used here by performing the reaction in solution under cry- tially covers the solvent accessible P1 active site. Here a com- oprotection conditions (Fig. 4A). As expected, no absorbance parison with the B. subtilis PDX1/PDX2 complex (in which the corresponding to the formation of the product PLP (414 nm) substrate is absent) and substrate bound P. berghei PDX1 was observed, due to the absence of the second substrate G3P. revealed that α2′ of PDX1.3 is in the inward conformation de- The structure of PDX1.3 (PDX1.3-adduct) under these condi- spite the absence of substrate (Fig. 3B). In an analogous fashion, tions was solved to 1.8 Å (Rwork, 18.0%; Rfree, 20.4%; Table S1) another short helix, α8′, adopts an outward and inward position and contains the same resolvable areas as the apoenzyme, with over the P1 site dependent on the absence or presence of sub- the addition of N297 that is resolved in this structure. The strate, respectively (11, 18, 32). Here we observe that this helix is global architecture of the PDX1.3-adduct obtained remained in the inward conformation in PDX1.3 (Fig. 3 A and C), despite very similar to that observed in the absence of added substrate the absence of substrate. The dipole of this short helix contrib- (rmsd 0.311 Å comparing 271 Cα atomsoverthetwostruc- utes to coordination of bound sulfate in PDX1.3 (equivalent to tures) (Fig. 4B). Nonetheless, key differences were observed in the phosphate of bound substrate). Additionally, a serine residue the P1 site in particular. First, the essential catalytic aspartate

Robinson et al. PNAS Early Edition | 3of9 Downloaded by guest on October 1, 2021 residue (D40 in PDX1.3) in P1 (10) that resides on the β1- strand and is oriented toward the center of the P1 site is shifted 1.0 Å deeper into the active site (and toward the adduct mol- A α8'' ecule; see below) compared with its position in the apo-PDX1.3 structure (Fig. 4C). This advanced position of D40 in the α8' α8' α8'' α2' presence of substrate represents a conformation either poised to perform or performing catalytic attack, consistent with its pre- α2' dicted role in shuffling protons during the formation of the chromophoric adduct (10). In the related structure of PDXS from G. stearothermophilus (Chain F; 4WY0), the catalytic as- partate does not adopt such an advanced position. Second, a dramatic reorientation is observed with the second catalytic ly- C-terminal sine residue, K165, which pointed toward the P2 site in the region C-terminal apostructure but has rotated through 120° to position itself in the region P1 site in the adduct structure (Fig. 4C). The precise role and mechanism of this lysine residue has been a matter of debate (4, 10, 17). Nevertheless, it is hypothesized that this residue couples the P1 and P2 active sites by a swinging arm mechanism pivoting between the two sites. However, with the exception of the most B β2 C recently described structure of the G. stearothermophilus en- zyme (15), it has been exclusively observed oriented toward the β1 P2 site, regardless of the presence or absence of substrate β3 (R5P) or product (PLP) in both prokaryotic and eukaryotic structures (11, 15, 17, 18, 32). Thus, orientation of this lysine residue toward the P2 site presumably represents the resting A66 S252 α8' state. Although the rotated position of the equivalent catalytic 3.1Å lysine has been observed recently in the G. stearothermophilus enzyme (15), the heterogeneous nature of the P1 site with re- α2' α2' spect to the ligand obscured its function. We observed consis- tent additional density indicating a covalently bound molecule to the catalytically essential K97 residue in all P1 sites in the D PDX1.3-adduct structure. The chemical structure of the chro- G258 mophoric intermediate proposed by Hanes et al. (8) from NMR studies can be modeled into this density. The initial imine D79 formed between K97 and R5P occurs at C1 of the latter R263 α8'' α2 (Fig. 1), but it has been proposed that this migrates to C5 during α8' the course of the formation of the chromophore (8). However, G253 the adduct observed here is poorly described by the electron R76 density when oriented so as to bond to K97 via C5 (Fig. 4D). In addition, the mean Bfactor for the adduct in this orientation is V74 2 α2' high (76.0 Å ). By contrast, when oriented to bind K97 via α2' T170 C1, the chromophore is described more clearly, albeit not completely, by the electron density (Fig. 4D) and has a reduced 2 Bfactor (58.8 Å ). A model of the same intermediate in a structure of the G. stearothermophilus enzyme proposed that the Fig. 3. Arabidopsis apo-PDX1.3 adopts a catalytically poised structure. (A) Neighboring subunits on the same hexameric ring. In apo-PDX1.3, struc- adduct is bonded via C5 [although the proposed chromo- tural elements α2′ and α8′ (blue) are located at the top of the P1 site, and phoric intermediate was observed in only one subunit of the the C-terminal region (blue) extends across the subunit interface. (B)Two dodecamer (15)]. Significantly, the model of the proposed chromo- conformations can be distinguished for the α2′ helix. An outward con- phore in the electron density present in the P1 site fits very well to a formation is seen in the ternary PDX1/PDX2 complex from B. subtilis species covalently bound to both K97 and K165 (Fig. 4D). It is (2NV2; dark gray), which is in the absence of the R5P substrate. By contrast, tempting to suggest that this represents a transfer intermediate and an inward conformation is seen in the structure of P. berghei solved in the thus captures the mechanism by which this reaction intermediate is presence of R5P (4ADU; light gray). The α2′ helix in Arabidopsis PDX1.3 transferred from the P1 to the P2 site; that is, K165 binds the adduct adopts an inward conformation despite the absence of substrate (green). via C5 in preparation for transfer to the P2 site. This would not (C) In an analogous fashion, the α8′ helix can adopt either an outward conformation, as seen in the absence of R5P in the ternary complex of necessitate the C1 to C5 migration proposed by Hanes et al. (8), PDX1/PDX2 complex from B. subtilis (2NV2; dark gray), or an inward con- which was deduced from highly processed enzyme that had been formation, as seen in the P. berghei PDX1 in the presence of R5P (4ADU; treated with acid and denatured with urea. Although this species has light gray). Arabidopsis PDX1.3 adopts the inward conformation despite not been observed in mass spectrometry (MS) data (10, 15), it should the absence of R5P and interacts via S252 with the N-terminal end of the be noted that such a modification would not affect the subunit intact α2′ helix (green) through its dipole and A66. (D) The C-terminal region mass as determined by electrospray ionization–MS but may not be (green) occupies a position in the lumen at the interface with a neigh- amenable to tryptic digestion and/or analysis by matrix assisted laser boring subunit (light green). It coordinates interactions with both subunits desorption/ionization–time of flight–MS. Notably, the electron den- zippering it in place. On one side, a series of hydrogen bonds comprising sity for the observed adduct is distinct from that of the anion bound the guanidinium group of R263 and amide groups of G253, G258, and T170 within the same subunit coordinate the peptide backbone of the to the phosphate binding loop (Fig. 4D). This observation validated C-terminal region directly or through coordination of waters (cyan). This is the processing of R5P and demonstrated the catalytic competence matched on the other side, by interaction with the side chains of R76, D79, of PDX1.3 in the crystal. Additional density adjoining the adduct, and the amide of V74 located in the loop connecting α2′ and α2ofthe but not describing the chromophore itself, should be noted. Its adjacent subunit, in addition to a network of structural waters. presence may indicate multiple isomeric species, possibly as a result

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1608125113 Robinson et al. Downloaded by guest on October 1, 2021 A B PNAS PLUS 310 nm 0.085

0.065 0 min P1 site 5 min 0.045 15 min 30 min 60 min P2 site 0.025 90 min 414 nm Absorbance (AU) Absorbance 0.005

250 300 350 400 450 500 Wavelength (nm) C D D40 β1 D40 D40 1Å

K97 β3 K165 K165 K165 120º K97 K97 54º β6 T164

Fig. 4. Capture of the swinging arm lysine in the structure of PDX1.3 containing a chromophoric intermediate. (A) Formation of the chromophoric intermediate by PDX1.3 in the presence of R5P in solution under cryoprotection conditions. A typical increase in absorbance at 310 nm over time in the presence of substrate BIOCHEMISTRY and an ammonium source (ammonium sulfate from the crystallization solution) is observed. Difference spectra of PDX1.3 (74 μM) in the absence and presence of R5P (6 mM) are shown. (B) The overall architecture of the apo-PDX1.3 (green) and PDX1.3-adduct (beige) structures are very similar. The catalytically important lysine (K165) that pivots between the P1 and P2 sites upon comparison of both structures is indicated. (C) A close-up view of the comparison of the P1 and P2 active sites in apo-PDX1.3 (green) and PDX1.3-adduct (beige) to indicate the inward movement of the catalytically important residue, D40, by 1 Å, as well as the

pivoting through 120° of K165. Note the reorientation of the neighboring threonine residue (T164) by 54°. (D) Fo–Fc omit map (2.5 σ) of the proposed structure of the chromophoric intermediate (yellow), K97 (beige), K165 (beige), and bound anion (gray). Left shows that of the proposed chromophore covalently bound to K97 via C5, whereas Right shows the chromophore bound to K97 via C1. The electron density matches well with a transfer intermediate, where C5 of the chromophore is bound to K165. Note the density for the bound anion is distinct from that of the covalently bound species.

of sample processing. A similar effect may account for the presence sequence of reactions that process covalently bound R5P to the of the chromophore in just 1 of the 12 subunits in a structure of chromophoric adduct because in the presence of unprocessed R5P G. stearothermophilus PDX1 (15). Taken together, the comparison this lysine residue points into P2. of the apo-PDX1.3 and the PDX1.3-adduct structures presented A notable barrier to the swing required by K165 to adopt the here clearly defined a consistent P2 site orientation for K165 in the P1 site orientation is a salt bridge formed between E121 and absence of substrate and P1 site orientation in the presence of the R163 (Fig. 5C). The proximity and pseudoplanar nature of this chromophoric adduct. Therefore, we have captured the two key salt bridge indicate that it is a strong interaction, and it is unlikely snapshots of the swinging arm mechanism of this lysine residue that passage of K165 to the P1 site would break it. The corre- originally postulated by Ealick and coworkers (17). sponding residue in B. subtilis PDX1 is a glutamate also (E105), and its mutation to an aspartate increased the specificity constant Mechanistic Insight into the Lysine Swinging Arm of PDX1.3. Further for formation of the chromophoric adduct approximately twofold examination of the apo- and PDX1.3-adduct structures allowed us (12). It was thus proposed that this salt bridge acts as a steric gate to provide mechanistic insight into the movement of K165 from the in coordination of P1 and P2 site activities (12). It is possible that P2totheP1site(Fig.5).WhenorientedtowardP2(restingstate), the smaller aspartate residue would move the impeding salt bridge the amide group of K165 hydrogen bonds with the peptide back- away from the P2 site-oriented lysine, increasing maneuverability bone of C145 (Fig. 5A). In this orientation, the hydroxyl group of of this catalytic residue. It therefore seems likely instead that the the T164 residue neighboring K165 hydrogen bonds with the car- trajectory of the K165 swing is through the opening that is lined bonyl group of A228 on β7, and its peptide backbone interacts with with P65, V122, and A168 and moreover may serve as a hydro- R163 (on β6; Fig. 5A). In the PDX1.3-adduct structure, the amide phobic gate between the active sites (Fig. 5C, Left and Right). nitrogen of K165 retains its interaction with C145, but the e-amino Although the hydrophobic nature of this gate would seem to act as group is reoriented 120° from P2 to interact with the chromophoric an obstacle to the swing from the P2 site, it may also serve a adduct in P1, allowing the carbonyl of K165 to hydrogen bond with ratchet-like function, preventing return of K165 to the P2 site until H179 (Fig. 5B). In parallel, the hydroxyl group of T164 hydrogen its action is complete and further energy is provided—possibly bonds with Q225, which has adopted a different rotamer position through binding of the second substrate G3P. to satisfy this interaction, and the amide carbonyl has rotated to Remarkably, despite substantial reorientation of K165 and form a hydrogen bond with A228 in the neighboring β-strand (β7). T164 between the apo-PDX1.3 and PDX1.3-adduct structures, It should be noted that Q225 retains two hydrogen bond interac- the conformational changes in this region are primarily restricted tions in both structures (Fig. 5 A and B), and overall conservation to these two residues. This stability is due, in part, to the of hydrogen bonds following formation of the chromophoric in- neighboring glycine residue, G166 (Fig. 5 A and B), which is able termediate indicates that the local rearrangement of this region is to tolerate large deviations in backbone torsion angles (33). A stable. Presumably, these conformational changes are driven by the comparison with the recent G. stearothermophilus structures (15),

Robinson et al. PNAS Early Edition | 5of9 Downloaded by guest on October 1, 2021 ABbonds with the peptide backbone of A168, stabilizing the loop. K97 K97 Therefore, the α2′ helix acts as a catch, restricting movement in β5 β5 the loop connecting the β6 strand and the α6helix.Theα2′ helix P1 site P1 site R163 is disordered in the matching structure of the G. stearothermophilus R163 β6 enzyme, and so movement of the corresponding loop is not E121 β6 A228 restricted in this species. Third, in PDX1.3 a glutamate (E167) A228 E121 K165 β7 located in the loop forms hydrogen bonds with T170 and coor- K165 β7 P2 site dinates a water molecule with the peptide backbone of a region P2 site Q225 C145 Q225 G244 C145 G166 near the C terminus of the protein (Fig. 6C). This glutamate pin T164 G166 T164 G244 provides additional stabilization to the loop connecting the β6 H179 H179 N184 strand and the α6 helix. The C-terminal region of the matching α6 N184 α6 G. stearothermophilus structure is not resolved; presumably, it is disordered and unable to form hydrogen bond interactions. C Consequently, there is no hindrance to movement of the corre- α2' sponding glutamate: it adopts a rotated position and provides no α2' stabilization of the loop (Fig. 6C). The structural rigidity con- ferred on the Arabidopsis PDX1.3 protein in the absence of P65 P65 P1 site A168 substrate and in the presence of the adduct is further emphasized P1 site V122 by the Bfactor values, which indicate a decrease in thermal motion V122 K165 A168 in the adduct structure (Fig. 6D and Table S2). All of this K165 combined serves to illustrate not only the pathway of reor- 90º R163 E121 ientation of K165 from P2 to P1 but also the stabilized assembly R163 of key structural elements required from a catalytic poised to an operational PDX1.3.

P2 site E121 β6 Substrate Access to the P1 Site. An open question is how the β6 P2 site substrate R5P entered the catalytically primed state of the plant enzyme because the defined geometric rearrangements have Fig. 5. Defining the K165 swinging arm in PDX1.3. Close-up views of the previously been postulated to represent a closed state to sub- interactions between K165 and surrounding residues in (A) apo-PDX1.3 strate (11, 15). Specifically, in previous structures of PDX1, more (green) and (B) PDX1.3 adduct (beige). Nonspecific secondary structure in- open conformations are defined in the absence of substrate, in teractions have been omitted for clarity. K165 is stabilized in a P2 orienta- which α2′ is in the outward conformation or disordered and the tion through interactions with the peptide backbone of C145, in which the hydroxyl group of T164 hydrogen bonds with the carbonyl group of A228, C-terminal region is disordered also, and thus, the P1 site is β accessible from the lumen of the dodecamer via a water filled and its peptide backbone interacts with R163 and the adjacent strand 7. – K165 P1 orientation is stabilized by a hydrogen bond interaction between its channel (11, 15 18, 29, 32). However, in the Arabidopsis apo- carbonyl group and H179. In this orientation, the C145 interaction is main- PDX1.3 structure, the inward conformation of α2′ and α8′ and tained, but the hydroxyl group of T164 hydrogen bonds with Q225, which the ordered conformation of the C-terminal region block access has adopted a different rotamer position, and the amide carbonyl has ro- to the P1 site via this route (Fig. 7A). We note here that the apo- tated to form a hydrogen bond with A228 in β7. (C) A salt bridge formed PDX1.3 has a second, water-filled cavity, located at the surface between E121 and R163 forces the swing of K165 from the P2 to the P1 site of the enzyme above the P1 site that extends down to K97 (Fig. through the opening lined with P65, V122 and A168 (compare Left and 7 A and B). It is plausible that R5P can enter via this cavity, and Right, oriented 90° from each other along the axis indicated). moreover, in forming the Schiff base with K97, the alkyl side chain of this residue would block the end of the cavity (Fig. 7B). where the active site lysine is also seen in both orientations, indicates Furthermore, a comparison of the apo-PDX1.3 structure with β α that there is much greater structural rigidity in the Arabidopsis that of the PDX1.3-adduct reveals that 1, 1, and the inter- enzyme than in its bacterial counterpart. This rigidity arises connecting loop have dropped down toward the P1 site (Fig. 7C). from three key differences: First, in the structure of the As well as moving D40 into position for catalytic attack, this shift G. stearothermophilus protein with the equivalent active site ly- has the effect of closing the described cavity. Valine 42, located at the apex of the β1/α1 loop, is moved into proximity to the peptide sine (K149) pointing into the P2 site, a proline residue (P152) carbonyls of L61 and E62, located on the loop connecting β2 adopts a position oriented in toward the P1 site (Fig. 6A). Fol- and α2′. To compensate for this unfavorable interaction, this lowing the swing of K149 from the P2 site to the P1 site, the second loop is turned away, and the side chain of E62 twists to unfavorable interaction that would result from positioning the e cover the mouth of the cavity, where it forms a salt bridge with -amino group of this residue 2.7Å away from the proline is the guanidinium group of R63 and hydrogen bonds with the circumvented by reorientation of P152 away from the lysine (Fig. amide nitrogen of R76 and a water, coordinated by polar and 6A). The reduced torsional flexibility of a proline residue means charged groups, located at the entrance of the proposed cavity the polypeptide backbone moves to accommodate its shifted (Fig. 7 C and D). It is possible that this gating mechanism orientation (Fig. 6A). In Arabidopsis PDX1.3, an alanine (A168) prevents substrate access to the P1 site before completion of occupies the equivalent position of P152 in G. stearothermophilus a round of PLP biosynthesis. Significantly, a similar cavity is PDX1, and therefore, its smaller side chain does not provide the observed in all other structures of PDX1, which although same obstruction. Consequently, repositioning of this loop in the slightly variable in the surface starting point all run down to the Arabidopsis PDX1.3 structure is not required (Fig. 6A). Second, active site lysine (Fig. 7E). Therefore, this cavity is likely to the loop region connecting the β6 strand and the α6 helix in serve as an access point for R5P, particularly in organisms that PDX1.3 is stabilized by interactions with the α2′ helix (Fig. 6B). use this catalytically poised configuration of the enzyme. Fur- The first turn of α2′ contains a proline (P65) and an alanine (A66), thermore, the access cavity itself mirrors the channels in which form a hydrophobic pocket with residues A168 and G169, aquaglyceroporins (34) and lactose permease (35, 36), in terms located at the top of the loop. The second turn of α2′ contains an of the residues present (conserved arginine at entrance with arginine (R69), the guanidinium group of which forms hydrogen some hydrophobic surfaces).

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1608125113 Robinson et al. Downloaded by guest on October 1, 2021 PNAS PLUS A BCα2' C-terminal A168 region 2.7 Å T170 P152 P65 A66 R69 P1 site G169 K165 A168 E151 K165 E167 K165 β6 P2 site α6 β6 β6 α6 α6 K149 D

α8' α8'

α2' α2'

C-terminal α6 region α6 C-terminal region 14.3 Å2 64.6 Å2

Fig. 6. Conformational stability of the PDX1.3-adduct structure parallels a catch and pin. (A–C) Three features define conformational stability in the Ara- bidopsis PDX1.3-adduct (beige) upon comparison with its bacterial counterpart from G. stearothermophilus (cyan and gray). A proline residue, P152, in the BIOCHEMISTRY G. stearothermophilus enzyme is displaced by the P2 site lysine (K149) upon formation of the chromophoric adduct [compare gray (1ZNN) and cyan (4WY0) depictions, before and after formation of the chromophore, respectively]. Consequently, the path of the loop connecting β6 and α6 deviates from the ground state position during catalysis in this species. The proline residue is an alanine in Arabidopsis (beige), and so a deviation is not required upon formation of the chromophoric adduct (A). The loop region connecting the β6 strand and the α6 helix in Arabidopsis PDX1.3 is stabilized by interactions with the α2′ helix, which acts like a catch (B). Movement of the loop is restricted by P65 and A66 in the first turn of α2′ forming a hydrophobic pocket with A168 and G169, located at the top of the loop. The guanidinium group of R69 in the second turn of α2′ forms a hydrogen bond with the peptide backbone of A168, further stabilizing the loop. The α2′ helix is disordered in the matching structure of the G. stearothermophilus enzyme, and so movement of the corresponding loop is not restricted in this species. A glutamate residue (E167) in Arabidopsis PDX1.3 (beige) located in the loop connecting β6andα6 forms hydrogen bonds with T170 and coordinates a water molecule with the peptide backbone of a region near the C terminus of the protein (C). This glutamate pin provides additional stabilization to this loop. The C-terminal region of the matching G. stearothermophilus structure is not resolved, and there is no hindrance to movement of

the corresponding glutamate: it adopts a rotated position providing no stabilization of the loop. (D) Bfactor coloration of the apo-PDX1.3 (Left) and PDX1.3- adduct structures (Right). An increase in thermal motion is indicated by a shift in color from blue to red.

Conclusion reactions can be conducted by a single enzyme with no need to re- The data presented here provide a structural view of PLP bio- cruit distinct partners or remodel an active site. synthesis de novo in plants. They reveal that in the absence of In summary, we have captured key snapshots of the plant PDX1 substrate, PDX1.3—in contrast to its bacterial counterparts—adopts in action, helping to define the intricate workings of a highly a catalytically poised conformation, in which several structural fea- complicated enzyme, essential for plant survival and important as tures around the P1 active site are in place. A water-filled cavity is a source of vitamin B6 to animals including humans. observed above the catalytic lysine (K97) of the P1 site, which is plausible to allow the entry of R5P in the Arabidopsis enzyme. In the Methods presence of the pentose phosphate substrate, an intermediate cor- Protein Expression and Purification. The construct pET–PDX1.3 containing the responding to the predicted chromophoric adduct molecule at the PDX1 homolog from Arabidopsis designed for expression of the protein with P1 site is observed, and the mouth of the cavity is closed off by a C-terminal hexa-histidine tag as described previously (37) was used in this study. Expression in E. coli BL21 (DE3) RIL cells was induced by the addition of reorientation of a glutamate residue. In this PDX1.3-adduct struc- 1 mM IPTG, followed by growth for 6 h at 28 °C (30). For protein purification, ture, the catalytic aspartate, D40, is in an attacking conformation, the cell pellet from 1 L of expression culture was resuspended in 100 mM extended toward the adduct at the center of the P1 site. Significantly, sodium phosphate buffer, pH 7.5, containing 300 mM NaCl, 10 mM imid- K165 previously thought to coordinate the P1 and P2 active sites has azole, 1 mM β-mercaptoethanol, and 1 mM PMSF (Buffer A). Lysozyme pivoted from its resting position in the P2 site to the P1 site and (0.3 mg·mL−1), DNaseI (50 μg·mL−1), and a protease inhibitor mixture (EDTA-free; provides a clear demonstration of a swinging arm mechanism. A Roche) were added to the cell suspension, and lysis was performed by son- lysine-mediated swinging arm mechanism has been described pre- ication. Insoluble material was removed by centrifugation (18,000 × g for 30 min viously for a variety of different protein families, including Class I at 4 °C), and the clarified lysate was applied to a gravity flow column con- aldolases (21), 2-oxoacid dehydrogenases, and the H protein of taining 0.5 mL nickel-nitrilotriacetic acid affinity resin (Macherey–Nagel) glycine decarboxylases (reviewed in ref. 28). However, they differ preequilibrated with Buffer A. Lysate was applied to the column a total of three times, and bound material was washed with 25 mL Buffer A followed from that described here in that they either occur within a single by 25 mL of Buffer A containing 50 mM imidazole. Protein was eluted from active center (class I aldolases) or mediate shuttling of coenzyme the column with Buffer A containing 300 mM imidazole and concentrated to bound intermediates. PDX1 makes use of the lysine swinging arm to 500 μL with partial buffer exchange (1:1) to 20 mM Tris·HCl, pH 7.0, con- coordinate distal divergent reaction sites, facilitating enzyme chem- taining 200 mM potassium chloride and 10 mM DTT followed by application istry that notably has no need for a coenzyme intermediate. This thus to a preequilibrated Superdex200 (10/300; GE Healthcare) at 0.4 mL·min−1. provides an elegant example of how a sequence of biochemical Fractions containing the dodecameric form of PDX1.3 (30) were collected

Robinson et al. PNAS Early Edition | 7of9 Downloaded by guest on October 1, 2021 A

2 1 2 1 45º

B

45º 45º

K97 β3

β4 Lys-R5P C D α1

L61 V42

E62 E62 β1

R76 R63 D40 R76 R63

Lys-R5P E

P. berghei P. horikoshii G. stearothermophilus

Fig. 7. An alternative solvent-exposed cavity providing access to R5P. (A) Surface representation of B. subtilis PDX1 (gray, 2NV2; Middle) and Arabidopsis apo-PDX1.3 (green; Right), overlaid on their respective chain representations. Positively charged and negatively charged residues are colored blue and red,

respectively. The orientation of the molecule relative to the typical depiction of the (β/α)8 barrel is shown (Left). The bacterial enzyme possesses two solvent exposed cavities, labeled 1 and 2. The poised conformation of apo-PDX1.3 obscures cavity 1, but cavity 2 is open to solvent. (B) Close-up of the water-filled access channel (Middle) proposed to be the site of R5P entry in apo-PDX1.3 (site 2). Coloring as in A. Right illustrates the blocking of the channel by the alkyl group of the active site lysine following substrate binding and is overlaid by the covalently bound active site Lys-R5P intermediate (gray) from the P. berghei enzyme (4ADU) following alignment of the two structures. (C) A comparison of apo-PDX1.3 (green) and PDX1.3-adduct (beige) illustrates movement of β1, α1, and E62 in Arabidopsis PDX1.3 following formation of the chromophoric adduct. The Lys-R5P intermediate from P. berghei is depicted (gray) as in B, Right.As well as moving D40 into position for catalytic attack (short arrow), this shift has the effect of closing the described cavity. V42, located at the apex of the β1/α1 loop, is moved into proximity to the peptide carbonyls of L61 and E62, located on the loop connecting β2 and α2′. To compensate for this unfavorable in- teraction, this second loop is turned away, and the side chain of E62 twists to cover the mouth of the cavity, where it forms hydrogen bonds with the guanidinium group of R63, the amide nitrogen of R76, and a water, coordinated by polar and charged groups, located at the entrance of the proposed cavity (D). In D, E62 from the PDX1.3-adduct structure is superimposed on the apo-PDX1.3 structure as shown in B.(E) A similar site 2 cavity is observed in all other structures of PDX1 as illustrated for P. berghei (4ADU), P. horikoshii (4FIR), and G. stearothermophilus (4ZXY).

and analyzed by SDS/PAGE, and protein concentration was measured by replaced in a stepwise manner to obtain a final solution containing 20% infrared absorbance using the Direct Detect system (Merck Millipore). (vol/vol) ethylene glycol in 5% increments (3 min per step). Cryoprotected crystals were plunge-frozen in liquid nitrogen for storage and X-ray dif- Crystallization and Data Collection. Crystals were grown at 18 °C by sitting- fraction experiments. Crystals containing the chromophoric adduct were drop vapor diffusion. Crystallization drops, consisting of 5 μL protein solu- obtained by preparing crystals of apo-PDX1.3 as above, but cryoprotection tion (2 mg·mL−1) and 5 μL precipitant solution [100 mM Mes·OH, pH 6.5, solutions were supplemented with 6 mM R5P, and crystals were equilibrated containing 1.4 M ammonium sulfate and 10% (vol/vol) 1,4-dioxane], were for time intervals of 10 min to 27 h. A total of 22 diffraction datasets were equilibrated against a 1-mL reservoir of precipitant solution. Crystals with obtained from PXI at the Swiss Light Source (PSI) at a wavelength of 0.9786 rhombohedral morphology (space group H3; 146) and unit cell dimensions nm. Crystals of apo-PDX1.3 and PDX1.3-adduct diffracted to 1.46 Å and 178.5 Å × 178.5 Å × 115.8 Å appeared after 3–5 d and reached maximum 1.76 Å, respectively. For data collection statistics see Table S1. dimensions (100 μm × 50 μm × 50 μm) after 4 wk. Crystals were recovered with nylon loops (Molecular Dimensions) and transferred to crystallization solu- PDX1.3 Chromophoric Intermediate Formation. To mimic formation of the tion supplemented with 5% (vol/vol) ethylene glycol. This solution was previously described chromophoric intermediate in PDX1 (10, 38), formation

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1608125113 Robinson et al. Downloaded by guest on October 1, 2021 of the adduct was assessed in solution under cryoprotection conditions chromophoric intermediate were generated with JLIGAND (47). Validation PNAS PLUS using a 96-well microplate reader (Synergy2; BioTek). The stoichiometry of was performed with COOT (45), PDB_REDO (48), and MOLPROBITY (49), and the chromophore relative to total protein was estimated from the absor- structural analysis was performed with PYMOL and BAVERAGE (39). Re- bance maxima at 282 nm and 310 nm for PDX1.3 and chromophore, re- finement statistics are presented in Table S1. The resolvable region of the −1 −1 spectively, and the respective extinction coefficients of 5,960 M ·cm apostructure from Arabidopsis (residues 21–296, chain A) was aligned with −1 −1 (estimated from the PDX1.3 primary sequence) and 16,200 M ·cm (10). structures from S. cerevisiae (3O07, Chain A), B. subtilis (2NV2, Chain A), and G. stearothermophilus (4WXY, Chain A) (15, 16, 18). Alignment was per- Structure Solution and Refinement. Diffraction data were processed with formed with SUPER in PYMOL using Cα atoms and with no cycles of re- programs of the CCP4 suite (39). Diffraction images were analyzed and in- finement (50). Figs. 2–7 were prepared with PYMOL (50), and density maps tegrated with MOSFLM (40, 41), and structure factors were obtained with in Figs. 2 and 4 were prepared with PHENIX (51). SCALA (42). The PDX1.3 structure was determined by molecular replacement using the program PHASER MR (43) using a model prepared with CHAINSAW ACKNOWLEDGMENTS. We thank Vincent Olieric, Tomizaki Takashi, and all (44) based on the B. subtilis PDX1 homolog (2NV2). Phases for the chro- staff at the Swiss Light Source for assistance, as well as Stéphane Thore (Uni- mophoric intermediate were obtained using the apostructure as a search versity of Bordeaux) for critical analysis of structural refinements. Financial model. Model building and refinement were performed with COOT (45) and support is gratefully acknowledged from the Swiss National Science Founda- REFMAC5 (46), respectively, and coordinates and chemical restraints for the tion (Grant 31003A-141117/1 to T.B.F.) as well as the University of Geneva.

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