PNAS PLUS
Structural definition of the lysine swing in Arabidopsis thaliana PDX1: Intermediate channeling facilitating vitamin B6 biosynthesis Graham C. Robinsona, Markus Kaufmanna, Céline Rouxa, and Teresa B. Fitzpatricka,1 a
Department of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerland
Vitamin B6 is indispensible for all organisms, notably as the coenzyme form pyridoxal 5′-phosphate. Plants make the compound de novo using a relatively simple pathway comprising pyridoxine synthase (PDX1) and pyridoxine glutaminase (PDX2). PDX1 is remarkable given its multifaceted synthetic ability to carry out isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization, all in the absence of coenzymes or recruitment of specialized domains. Two active sites (P1 and P2) facilitate the plethora of reactions, but it is not known how the two are coordinated and, moreover, if intermediates are tunneled between active sites. Here we present X-ray structures of PDX1.3 from Arabidopsis thaliana, the overall architecture of which is a dodecamer of (β/α)8 barrels, similar to the majority of its homologs. An apoenzyme structure revealed that features around the P1 active site in PDX1.3 have adopted inward conformations consistent with a catalytically primed state and delineated a substrate accessible cavity above this active site, not noted in other reported structures. Comparison with the structure of PDX1.3 with an intermediate along the catalytic trajectory demonstrated that a lysine residue swings from the distinct P2 site to the P1 site at this stage of catalysis and is held in place by a molecular catch and pin, positioning it for transfer of serviced substrate back to P2. The study shows that a simple lysine swinging arm coordinates use of chemically disparate sites, dispensing with the need for additional factors, and provides an elegant example of solving complex chemistry to generate an essential metabolite. Arabidopsis thaliana lysine swing
| vitamin B6 biosynthesis | crystal structure |
zation, and aromatization to produce the PLP molecule has received considerable attention but remains enigmatic (7–10). Both biochemical and structural studies have served to capture snapshots of the PDX1 enzyme in action at certain stages along the catalytic trajectory (10–18). Nonetheless, the sequence of events cannot yet be stitched together and is essential to provide a complete overview of one of nature’s most complicated enzymes from a fundamental perspective, as well as its potential as a drug target. Multifunctional enzymes generally recruit distinct domains for their reactions, or connect different sites by substrate channeling (19, 20), or remodel a single active site (21) to facilitate the multitude of transformations taking place. Substrate channeling can be mediated by tunneling (22) or by transfer via a covalently bound prosthetic group to the successive active site. Classic examples of the latter are lipoyl-lysine residues of 2-oxo acid dehydrogenases (23), biotinyl-lysine residues of carboxylases (24), and phosphopantetheinyl-serine residues of fatty acid synthases (25, 26). Notably, the referred to prosthetic groups are all vitaminderived coenzymes. The covalently attached prosthetic groups generally have freedom to rotate within their respective active sites and thus serve as swinging arms ferrying substrates or intermediates to the subsequent active site (27). Enhancement of catalytic efficiency, channeling, and protection of stable intermediates are hypothesized reasons for such approaches (28). Nonetheless, the concept has remained open to questions, which include whether the decorated side chain swings or the whole protein domain, as well as the need for the posttranslational Significance
V
itamin B6 in its form as pyridoxal 5′-phosphate (PLP) is an essential coenzyme involved in over 140 biochemical reactions, more than any other known nutrient. It is the most versatile coenzyme known in nature being involved in transamination, decarboxylation, elimination, racemization, and replacement reactions (1). It is biosynthesized de novo by microorganisms and plants. As an essential micronutrient, it must be taken in the diet of animals including humans. The absence of the pathway in animals renders the biosynthesis de novo pathway a potential drug target in pathogenic organisms (2). It is now established that there are two biochemical routes that lead to the biosynthesis de novo of PLP. The first to be resolved was the deoxyxylulose 5-phosphate (DXP)-dependent pathway, which requires seven enzymes and is found in a few select microorganisms including Escherichia coli (reviewed in ref. 3). The second route does not involve DXP (DXP-independent), only requires two enzymes, and is by far the predominant route being found in most microorganisms and all plants (reviewed in ref. 3). The two enzymes required for the DXP-independent route are pyridoxine synthase (PDX1) and pyridoxine glutaminase (PDX2), which together form a glutamine amidotransferase complex that uses ribose 5-phosphate (R5P), glyceraldehyde 3-phosphate (G3P), and glutamine in a highly complicated sequence of reactions to produce PLP (4, 5) (Fig. 1). Although PDX2 is a classic glutaminase hydrolyzing glutamine to ammonia and glutamate (4–6), the remarkable reaction mechanism of PDX1 that involves pentose and triose isomerization, imine formation, ammonia addition, aldol-type condensation, cycli-
www.pnas.org/cgi/doi/10.1073/pnas.1608125113
Multifunctional enzymes have been shown to recruit distinct domains for their reactions, remodel active sites, or connect different sites by substrate channeling to facilitate the multitude of transformations taking place. Pyridoxine synthase (PDX1) of the vitamin B6 biosynthesis machinery is a remarkable enzyme that alone has a polymorphic catalytic ability designated to two active sites, the coordination of which is unclear. Here structural snapshots allow us to describe a lysine swinging arm mechanism that facilitates serviced substrate transfer and demonstrates how an enzyme can couple distinct chemistry between active sites, dispensing with the need for extra domains, substrate tunneling, or transfer of coenzyme bound intermediates. The work provides an elegant example of simplicity at work in nature’s sea of complexity. Author contributions: G.C.R. and T.B.F. designed research; G.C.R. and M.K. performed research; G.C.R., M.K., and C.R. contributed new reagents/analytic tools; G.C.R. and T.B.F. analyzed data; and G.C.R. and T.B.F. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: Crystallography, atomic coordinates, and structure factors have been deposited in the RCSB Protein Data Bank [accession nos. 5K3V (apo-PDX1.3) and 5K2Z (PDX1.3-adduct)]. 1
To whom correspondence should be addressed. Email:
[email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1608125113/-/DCSupplemental.
PNAS | Published online September 19, 2016 | E5821–E5829
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Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved August 5, 2016 (received for review May 20, 2016)
OH
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Results and Discussion OPO32-
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Fig. 1. Scheme depicting selected reactions of PDX1 in the P1 and P2 active sites. Ribose 5-phosphate (R5P) enters the P1 active site of PDX1 and forms a covalent intermediate with K97 (Arabidopsis numbering). Loss of water and inorganic phosphate (Pi) from R5P and incorporation of ammonia (NH3) leads to formation of a covalent chromophoric adduct. The presence of K165 in the P1 site at this stage of catalysis facilitates transfer of the chromophoric adduct to the P2 active site. Condensation with glyceraldehyde 3-phosphate (G3P) to form the product pyridoxal 5′-phosphate (PLP) completes one catalytic cycle. The interactions of the e-amino groups of the respective lysines with either R5P or the derived chromophoric adduct are depicted in gray.
modification to include the prosthetic group facilitating the multistep chemical reaction (28). The complex chemistry carried out by PDX1 provides an intriguing example to investigate in the context of multifunctional enzymes given that no prosthetic group is required and the enzyme alone carries out the entire series of reactions. All PDX1s are made up of two active sites referred to as P1 and P2 (16, 17, 29) and together coordinate the sequence of reactions resulting in PLP production (Fig. 1). The process begins with the binding of R5P in the P1 site in a covalent interaction with an essential lysine residue [K97 in Arabidopsis thaliana (hereafter referred to as Arabidopsis) PDX1.3] (10, 17). Loss of phosphate and water from R5P as well as the incorporation of ammonia (from glutamine in the presence of PDX2) result in a chromophoric intermediate likely to be unique to PLP synthase, which has been observed spectroscopically (8–10) but has eluded precise structural characterization. The reaction can only proceed further in the presence of G3P, which is assumed to take place in the P2 site and is where the final product PLP has been observed in X-ray crystal structures (10, 12, 18). A pertinent question is how the intermediate is transferred from the P1 to the P2 site. A second lysine (K165 in Arabidopsis PDX1.3) is postulated to orchestrate the interaction between the active sites (10, 17) but had only been observed pointing toward the P2 site until recently. The recent report captured PDX1 in a rather heterogeneous population of different catalytic states in which this second lysine points into either the P1 or P2 site (15); however, the mixture of states in each subunit precluded clarification of the transition between the two states. Precise definition of the swinging of this essential lysine between the active sites would help to resolve the obscure steps between the initiation of the PLP synthase reaction in P1 and its completion in P2. Here we have determined X-ray crystal structures of a PDX1 (PDX1.3) from Arabidopsis. Like its bacterial counterparts, the plant protein displays dodecameric architecture. In contrast to previous structures, the Arabidopsis apoenzyme is in a closed conformation but poised for catalysis and serves to delineate a cavity above the P1 active site that provides access to the R5P substrate. In the apoenzyme, the second active site lysine is oriented toward the P2 site defining the resting state, i.e., its position at the beginning and end of the catalytic cycle. The structure of Arabidopsis PDX1.3 with the chromophoric intermediate solved to 1.8 Å was also determined and can be used to corroborate a proposed structure for this adduct. Moreover, this structure clearly placed the second active site lysine in the P1 site at this stage of E5822 | www.pnas.org/cgi/doi/10.1073/pnas.1608125113
catalysis. A molecular catch and pin mechanism explains the restraint of this lysine in P1, release of which presumably facilitates the swing back to P2 and completion of PLP biosynthesis. Overall, these snapshots of the plant PDX1 provide key insights into the intricate workings of a remarkable enzyme essential for plant survival and more generally into multifunctional enzymes. PDX1.3 from Arabidopsis Adopts a Dodecameric Architecture. The
structure of apo-PDX1.3 from Arabidopsis has been determined at 1.9 Å resolution (Rwork, 17.7%; Rfree, 20.3%; Table S1). It comprises residues 20–296 with 13 residues not visible at the C terminus in addition to the 19 missing from the N terminus. Twelve PDX1.3 subunits that fold as (β/α)8 barrels form a double hexameric ring interdigitating into the dodecameric structure (Fig. 2A). The dodecameric architecture is consistent with earlier estimations of the molecular mass of PDX1.3 (411,000 Da) by size exclusion chromatography coupled to static light scattering (30). The individual hexameric rings are 100 Å in diameter with a central pore 40 Å in diameter (Fig. 2A). The two hexamers stack together to form a structure 90 Å high (Fig. 2A). The overall architecture is similar to homologs of PDX1.3 that have been crystallized from bacterial sources including Bacillus subtilis (2NV1 and 2NV2) (16), Geobacillus stearothermophilus (1ZNN, 4WXY, 4WXZ, and 4WY0) (15, 29), Thermotoga maritima (2ISS) (17), and Mycobacterium tuberculosis Rv2606c (4JDY) (31), as well as those from apicomplexan Plasmodium species (4ADS, 4ADT, and 4ADU) (11), all of which form dodecamers. Notably, structures of the homologous enzymes from the yeast Saccharomyces cerevisiae (3FEM, 3O05, 3O06, and 3O07) (13, 18) and the archaeon Pyrococcus horikoshii (4FIQ and 4FIR) (32) crystallize as hexamers. Labeling of individual α-helices and β-strands of PDX1.3 was done according to the labeling introduced for the B. subtilis homolog (Fig. 2B) (16). Thus, contact between each adjacent (β/α)8 barrel in PDX1.3 is established by helix α8′′, which runs parallel to helix α8 near the C terminus (Fig. 2 A and B), as for all homologs. As for other dodecameric PDX1s, the interdigitation of the hexameric rings in PDX1.3 is established by helices α6, α6′, and α6′′ of each subunit (Fig. 2 A and B). Notably, helix αN is present despite the absence of the glutaminase subunit. Previously, this helix has only been observed in complexes of PDX1 with PDX2, with the exception of PDX1 from S. cerevisiae (13, 16, 17). The P1 Active Site of apo-PDX1.3 Displays a Catalytically Poised Architecture. Two active sites, annotated P1 and P2, have been
definitively located in PDX1 enzymes (16, 17). These sites were originally defined by bound chloride, phosphate, or sulfate ions in X-ray crystallographic structures (16, 17, 29) and more recently by the bound substrate R5P (P1) and product PLP (P2) (11, 18, 32) and have been validated through several biochemical studies (10, 12, 16). In PDX1.3, the P1 site is located at the C-terminal mouth of the β-barrel and contains density for a sulfate ion surrounded by the characteristic phosphate-binding loop that includes Gly–Thr– Gly, as well as other established conserved residues of this active site (16, 29) (Fig. 2B). The P2 site is located at the interface between subunits of opposing hexamers and also contains density for a bound sulfate ion surrounded by the conserved residues characteristic of this region (Fig. 2B). As for the homologous counterparts of PDX1.3, the bound sulfates in P1 and P2 occupy the same position as the phosphates of bound R5P and PLP, respectively (11, 15, 17, 18, 32). Markedly, except for coordinated waters, there is no other density observed in the P1 or P2 sites, such that this structural snapshot of the PDX1.3 enzyme can be considered substrate and product free, i.e., apo-PDX1.3. Although the overall architecture of PDX1.3 from Arabidopsis is similar to that of its homologs (rmsd of 1.14 Å, 0.77 Å, and 1.35 Å comparing 250, 262, and 242 corresponding Cα atoms of Robinson et al.
40 Å
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Fig. 2. Overall architecture of PDX1.3 from Arabidopsis. (A) PDX1.3 forms a dodecamer composed of two interdigitated hexamers (green and light blue), shown from above and the side after rotation by 90°. Dimensions are as indicated. (B) The individual subunits of PDX1.3 adopt a (β/α)8 fold with additional structural elements labeled as indicated. The P1 and P2 catalytic sites can be clearly defined with residues characteristic of each site and sulfate ions (yellow and red sticks) shown. Note one residue in P2, lysine 203 (K203), is contributed by α6′ of a subunit on the opposing hexamer (light blue). Waters have been omitted for clarity. The electron density is contoured to 1.0σ.
B. subtilis, G. stearothermophilus, and S. cerevisiae PDX1, respectively), we observed that the small segment of each subunit inserted between β1 and α2 within the lumen of each hexameric ring, comprising residues 65–71, adopts an ordered conformation, α2′, in PDX1.3 from Arabidopsis (Fig. 3A). In the reported bacterial PDX1 structures, the α2′ fold has only been observed in structures of the ternary complex with the glutaminase PDX2 and was, moreover, suggested to result from priming by a signal from the glutaminase subunit (15–17). However, α2′ has since been observed in the reported structure of PDX1 alone from the archaeon P. horikoshii (4FIQ and 4FIR) (32), as well as in the eukaryotic PDX1 structures reported from S. cerevisiae (3FEM, 3O05, 3O06, and 3O07) (13, 18) and Plasmodium berghei (4ADT and 4ADU) (11), all of which were crystallized in the absence of PDX2. Furthermore, in the archaeal and eukaryotic structures, α2′ exists in either of two conformations (out or in), dependent on the absence or presence of R5P, respectively (11, 18, 32). In the presence of the substrate, the inward movement of α2′ partially covers the solvent accessible P1 active site. Here a comparison with the B. subtilis PDX1/PDX2 complex (in which the substrate is absent) and substrate bound P. berghei PDX1 revealed that α2′ of PDX1.3 is in the inward conformation despite the absence of substrate (Fig. 3B). In an analogous fashion, another short helix, α8′, adopts an outward and inward position over the P1 site dependent on the absence or presence of substrate, respectively (11, 18, 32). Here we observe that this helix is in the inward conformation in PDX1.3 (Fig. 3 A and C), despite the absence of substrate. The dipole of this short helix contributes to coordination of bound sulfate in PDX1.3 (equivalent to the phosphate of bound substrate). Additionally, a serine residue Robinson et al.
A Catalytically Operational PDX1.3 Captures a Lysine Swinging Arm Mechanism. To further test if the geometric arrangements observed
in PDX1.3 represent a catalytically poised state rather than a state closed to substrate (possibly caused by bound sulfate mimicking substrate binding), we performed crystal-soaking experiments with R5P. Indeed, it must be mentioned that the P1 site is predominantly hydrophobic, the majority of residue interactions only being with the phosphate moiety of the substrate. Because an ammonium salt is present in the crystallization solution, we anticipated that the enzyme would catalytically process R5P under these conditions. Specifically, we have shown previously that PDX1 can transform covalently bound R5P into a chromophoric intermediate with an absorption maximum around 315 nm that results from loss of water and phosphate in the presence of a source of ammonia (10) (Fig. 1). We verified that this intermediate forms under the crystallization conditions used here by performing the reaction in solution under cryoprotection conditions (Fig. 4A). As expected, no absorbance corresponding to the formation of the product PLP (414 nm) was observed, due to the absence of the second substrate G3P. The structure of PDX1.3 (PDX1.3-adduct) under these conditions was solved to 1.8 Å (Rwork, 18.0%; Rfree, 20.4%; Table S1) and contains the same resolvable areas as the apoenzyme, with the addition of N297 that is resolved in this structure. The global architecture of the PDX1.3-adduct obtained remained very similar to that observed in the absence of added substrate (rmsd 0.311 Å comparing 271 Cα atoms over the two structures) (Fig. 4B). Nonetheless, key differences were observed in the P1 site in particular. First, the essential catalytic aspartate PNAS | Published online September 19, 2016 | E5823
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located at the N terminus of this helix in PDX1.3, S252, forms a 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 observed that most of the C-terminal region (residues 286–296 out of 308) was resolved in PDX1.3 (Fig. 3A), even though the substrate 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 same hexamer (Fig. 3A). Remarkably, the peptide backbone of this region is zippered into place by intrasubunit and intersubunit interactions across this cleft (Fig. 3D). On one side, a series of hydrogen bonds comprising the guanidinium group of R263 and amide groups of G253, G258, and T170 within the same subunit coordinate the peptide backbone of the C-terminal region directly or through coordination of waters. This is matched on the other side by interaction with the side chains of R76 and D79 and the amide of V74 located in the loop connecting α2′ and α2 of the adjacent subunit, in addition to a network of structural waters (Fig. 3D). Taken together, the inward position of α2′ as well as α8′ and the coordination of the C-terminal region define three important structural arrangements that have only been collectively seen previously in PDX1 structures in the presence of the R5P substrate. As these arrangements serve to limit access to the P1 site, they are assumed to represent a catalytically operating state. However, we have observed this arrangement in the absence of substrate and must therefore conclude that this rather represents a catalytically poised state, at least in the case of PDX1.3. Moreover, the network of intrasubunit and intersubunit interactions zippering the C-terminal region in place would appear to be poised to coordinate cross-talk between two neighboring subunits. We have previously demonstrated that PDX1 displays high cooperativity in relation to the binding of the R5P substrate for which the C terminus is essential (12, 14). Therefore, this structure of PDX1.3 serves to capture the fundamental essence of the C terminus in linking neighboring catalytically poised subunits.
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Fig. 3. Arabidopsis apo-PDX1.3 adopts a catalytically poised structure. (A) Neighboring subunits on the same hexameric ring. In apo-PDX1.3, structural elements α2′ and α8′ (blue) are located at the top of the P1 site, and the C-terminal region (blue) extends across the subunit interface. (B) Two conformations can be distinguished for the α2′ helix. An outward conformation is seen in the ternary PDX1/PDX2 complex from B. subtilis (2NV2; dark gray), which is in the absence of the R5P substrate. By contrast, an inward conformation is seen in the structure of P. berghei solved in the presence of R5P (4ADU; light gray). The α2′ helix in Arabidopsis PDX1.3 adopts an inward conformation despite the absence of substrate (green). (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 PDX1/PDX2 complex from B. subtilis (2NV2; dark gray), or an inward conformation, as seen in the P. berghei PDX1 in the presence of R5P (4ADU; light gray). Arabidopsis PDX1.3 adopts the inward conformation despite the absence of R5P and interacts via S252 with the N-terminal end of the α2′ helix (green) through its dipole and A66. (D) The C-terminal region (green) occupies a position in the lumen at the interface with a neighboring subunit (light green). It coordinates interactions with both subunits zippering it in place. On one side, a series of hydrogen bonds comprising the guanidinium group of R263 and amide groups of G253, G258, and T170 within the same subunit coordinate the peptide backbone of the C-terminal region directly or through coordination of waters (cyan). This is matched on the other side, by interaction with the side chains of R76, D79, and the amide of V74 located in the loop connecting α2′ and α2 of the adjacent subunit, in addition to a network of structural waters.
E5824 | www.pnas.org/cgi/doi/10.1073/pnas.1608125113
residue (D40 in PDX1.3) in P1 (10) that resides on the β1strand and is oriented toward the center of the P1 site is shifted 1.0 Å deeper into the active site (and toward the adduct molecule; see below) compared with its position in the apo-PDX1.3 structure (Fig. 4C). This advanced position of D40 in the presence of substrate represents a conformation either poised to perform or performing catalytic attack, consistent with its predicted 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 aspartate does not adopt such an advanced position. Second, a dramatic reorientation is observed with the second catalytic lysine residue, K165, which pointed toward the P2 site in the apostructure but has rotated through 120° to position itself in the 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 recently described structure of the G. stearothermophilus enzyme (15), it has been exclusively observed oriented toward the P2 site, regardless of the presence or absence of substrate (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 state. Although the rotated position of the equivalent catalytic lysine has been observed recently in the G. stearothermophilus enzyme (15), the heterogeneous nature of the P1 site with respect to the ligand obscured its function. We observed consistent additional density indicating a covalently bound molecule to the catalytically essential K97 residue in all P1 sites in the PDX1.3-adduct structure. The chemical structure of the chromophoric intermediate proposed by Hanes et al. (8) from NMR studies can be modeled into this density. The initial imine formed between K97 and R5P occurs at C1 of the latter (Fig. 1), but it has been proposed that this migrates to C5 during the course of the formation of the chromophore (8). However, the adduct observed here is poorly described by the electron 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 high (76.0 Å2). By contrast, when oriented to bind K97 via C1, the chromophore is described more clearly, albeit not completely, by the electron density (Fig. 4D) and has a reduced B factor (58.8 Å 2 ). A model of the same intermediate in a structure of the G. stearothermophilus enzyme proposed that the adduct is bonded via C5 [although the proposed chromophoric intermediate was observed in only one subunit of the dodecamer (15)]. Significantly, the model of the proposed chromophore in the electron density present in the P1 site fits very well to a species covalently bound to both K97 and K165 (Fig. 4D). It is tempting to suggest that this represents a transfer intermediate and thus captures the mechanism by which this reaction intermediate is transferred from the P1 to the P2 site; that is, K165 binds the adduct via C5 in preparation for transfer to the P2 site. This would not necessitate the C1 to C5 migration proposed by Hanes et al. (8), which was deduced from highly processed enzyme that had been treated with acid and denatured with urea. Although this species has not been observed in mass spectrometry (MS) data (10, 15), it should be noted that such a modification would not affect the subunit intact mass as determined by electrospray ionization–MS but may not be amenable to tryptic digestion and/or analysis by matrix assisted laser desorption/ionization–time of flight–MS. Notably, the electron density for the observed adduct is distinct from that of the anion bound to the phosphate binding loop (Fig. 4D). This observation validated the processing of R5P and demonstrated the catalytic competence of PDX1.3 in the crystal. Additional density adjoining the adduct, but not describing the chromophore itself, should be noted. Its presence may indicate multiple isomeric species, possibly as a result Robinson et al.
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of sample processing. A similar effect may account for the presence of the chromophore in just 1 of the 12 subunits in a structure of G. stearothermophilus PDX1 (15). Taken together, the comparison of the apo-PDX1.3 and the PDX1.3-adduct structures presented here clearly defined a consistent P2 site orientation for K165 in the absence of substrate and P1 site orientation in the presence of the chromophoric adduct. Therefore, we have captured the two key snapshots of the swinging arm mechanism of this lysine residue originally postulated by Ealick and coworkers (17). Mechanistic Insight into the Lysine Swinging Arm of PDX1.3. Further examination of the apo- and PDX1.3-adduct structures allowed us to provide mechanistic insight into the movement of K165 from the P2 to the P1 site (Fig. 5). When oriented toward P2 (resting state), the amide group of K165 hydrogen bonds with the peptide backbone of C145 (Fig. 5A). In this orientation, the hydroxyl group of the T164 residue neighboring K165 hydrogen bonds with the carbonyl group of A228 on β7, and its peptide backbone interacts with R163 (on β6; Fig. 5A). In the PDX1.3-adduct structure, the amide nitrogen of K165 retains its interaction with C145, but the e-amino group is reoriented 120° from P2 to interact with the chromophoric adduct in P1, allowing the carbonyl of K165 to hydrogen bond with H179 (Fig. 5B). In parallel, the hydroxyl group of T164 hydrogen bonds with Q225, which has adopted a different rotamer position to satisfy this interaction, and the amide carbonyl has rotated to form a hydrogen bond with A228 in the neighboring β-strand (β7). It should be noted that Q225 retains two hydrogen bond interactions in both structures (Fig. 5 A and B), and overall conservation of hydrogen bonds following formation of the chromophoric intermediate indicates that the local rearrangement of this region is stable. Presumably, these conformational changes are driven by the Robinson et al.
sequence of reactions that process covalently bound R5P to the chromophoric adduct because in the presence of unprocessed R5P this lysine residue points into P2. A notable barrier to the swing required by K165 to adopt the P1 site orientation is a salt bridge formed between E121 and R163 (Fig. 5C). The proximity and pseudoplanar nature of this salt bridge indicate that it is a strong interaction, and it is unlikely that passage of K165 to the P1 site would break it. The corresponding residue in B. subtilis PDX1 is a glutamate also (E105), and its mutation to an aspartate increased the specificity constant for formation of the chromophoric adduct approximately twofold (12). It was thus proposed that this salt bridge acts as a steric gate in coordination of P1 and P2 site activities (12). It is possible that the smaller aspartate residue would move the impeding salt bridge away from the P2 site-oriented lysine, increasing maneuverability of this catalytic residue. It therefore seems likely instead that the trajectory of the K165 swing is through the opening that is lined with P65, V122, and A168 and moreover may serve as a hydrophobic gate between the active sites (Fig. 5C, Left and Right). Although the hydrophobic nature of this gate would seem to act as an obstacle to the swing from the P2 site, it may also serve a ratchet-like function, preventing return of K165 to the P2 site until its action is complete and further energy is provided—possibly through binding of the second substrate G3P. Remarkably, despite substantial reorientation of K165 and T164 between the apo-PDX1.3 and PDX1.3-adduct structures, the conformational changes in this region are primarily restricted to these two residues. This stability is due, in part, to the neighboring glycine residue, G166 (Fig. 5 A and B), which is able to tolerate large deviations in backbone torsion angles (33). A comparison with the recent G. stearothermophilus structures (15), PNAS | Published online September 19, 2016 | E5825
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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 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.
A
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Fig. 5. Defining the K165 swinging arm in PDX1.3. Close-up views of the interactions between K165 and surrounding residues in (A) apo-PDX1.3 (green) and (B) PDX1.3 adduct (beige). Nonspecific secondary structure interactions have been omitted for clarity. K165 is stabilized in a P2 orientation through interactions with the peptide backbone of C145, in which the hydroxyl group of T164 hydrogen bonds with the carbonyl group of A228, and its peptide backbone interacts with R163 and the adjacent strand β7. K165 P1 orientation is stabilized by a hydrogen bond interaction between its carbonyl group and H179. In this orientation, the C145 interaction is maintained, but the hydroxyl group of T164 hydrogen bonds with Q225, which has adopted a different rotamer position, and the amide carbonyl has rotated to form a hydrogen bond with A228 in β7. (C) A salt bridge formed between E121 and R163 forces the swing of K165 from the P2 to the P1 site through the opening lined with P65, V122 and A168 (compare Left and Right, oriented 90° from each other along the axis indicated).
where the active site lysine is also seen in both orientations, indicates that there is much greater structural rigidity in the Arabidopsis enzyme than in its bacterial counterpart. This rigidity arises from three key differences: First, in the structure of the G. stearothermophilus protein with the equivalent active site lysine (K149) pointing into the P2 site, a proline residue (P152) adopts a position oriented in toward the P1 site (Fig. 6A). Following the swing of K149 from the P2 site to the P1 site, the unfavorable interaction that would result from positioning the e-amino group of this residue 2.7Å away from the proline is circumvented by reorientation of P152 away from the lysine (Fig. 6A). The reduced torsional flexibility of a proline residue means the polypeptide backbone moves to accommodate its shifted orientation (Fig. 6A). In Arabidopsis PDX1.3, an alanine (A168) occupies the equivalent position of P152 in G. stearothermophilus PDX1, and therefore, its smaller side chain does not provide the same obstruction. Consequently, repositioning of this loop in the Arabidopsis PDX1.3 structure is not required (Fig. 6A). Second, the loop region connecting the β6 strand and the α6 helix in PDX1.3 is stabilized by interactions with the α2′ helix (Fig. 6B). The first turn of α2′ contains a proline (P65) and an alanine (A66), which form a hydrophobic pocket with residues A168 and G169, located at the top of the loop. The second turn of α2′ contains an arginine (R69), the guanidinium group of which forms hydrogen E5826 | www.pnas.org/cgi/doi/10.1073/pnas.1608125113
bonds with the peptide backbone of A168, stabilizing the loop. Therefore, the α2′ helix acts as a catch, restricting movement in the loop connecting the β6 strand and the α6 helix. 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. Third, in PDX1.3 a glutamate (E167) located in the loop forms hydrogen bonds with T170 and coordinates a water molecule with the peptide backbone of a region near the C terminus of the protein (Fig. 6C). This glutamate pin provides additional stabilization to the loop connecting the β6 strand and the α6 helix. The C-terminal region of the matching G. stearothermophilus structure is not resolved; presumably, it is disordered and unable to form hydrogen bond interactions. Consequently, there is no hindrance to movement of the corresponding glutamate: it adopts a rotated position and provides no stabilization of the loop (Fig. 6C). The structural rigidity conferred on the Arabidopsis PDX1.3 protein in the absence of substrate and in the presence of the adduct is further emphasized by the Bfactor values, which indicate a decrease in thermal motion in the adduct structure (Fig. 6D and Table S2). All of this combined serves to illustrate not only the pathway of reorientation of K165 from P2 to P1 but also the stabilized assembly of key structural elements required from a catalytic poised to an operational PDX1.3. Substrate Access to the P1 Site. An open question is how the substrate R5P entered the catalytically primed state of the plant enzyme because the defined geometric rearrangements have previously been postulated to represent a closed state to substrate (11, 15). Specifically, in previous structures of PDX1, more open conformations are defined in the absence of substrate, in which α2′ is in the outward conformation or disordered and the C-terminal region is disordered also, and thus, the P1 site is accessible from the lumen of the dodecamer via a water filled channel (11, 15–18, 29, 32). However, in the Arabidopsis apoPDX1.3 structure, the inward conformation of α2′ and α8′ and the ordered conformation of the C-terminal region block access to the P1 site via this route (Fig. 7A). We note here that the apoPDX1.3 has a second, water-filled cavity, located at the surface of the enzyme above the P1 site that extends down to K97 (Fig. 7 A and B). It is plausible that R5P can enter via this cavity, and moreover, in forming the Schiff base with K97, the alkyl side chain of this residue would block the end of the cavity (Fig. 7B). Furthermore, a comparison of the apo-PDX1.3 structure with that of the PDX1.3-adduct reveals that β1, α1, and the interconnecting loop have dropped down toward the P1 site (Fig. 7C). As well as moving D40 into position for catalytic attack, this shift 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 carbonyls of L61 and E62, located on the loop connecting β2 and α2′. To compensate for this unfavorable interaction, this second loop is turned away, and the side chain of E62 twists to cover the mouth of the cavity, where it forms a salt bridge with the guanidinium group of R63 and hydrogen bonds with the amide nitrogen of R76 and a water, coordinated by polar and charged groups, located at the entrance of the proposed cavity (Fig. 7 C and D). It is possible that this gating mechanism prevents substrate access to the P1 site before completion of a round of PLP biosynthesis. Significantly, a similar cavity is observed in all other structures of PDX1, which although slightly variable in the surface starting point all run down to the active site lysine (Fig. 7E). Therefore, this cavity is likely to serve as an access point for R5P, particularly in organisms that use this catalytically poised configuration of the enzyme. Furthermore, the access cavity itself mirrors the channels in aquaglyceroporins (34) and lactose permease (35, 36), in terms of the residues present (conserved arginine at entrance with some hydrophobic surfaces). Robinson et al.
2.7 Å
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Conclusion The data presented here provide a structural view of PLP biosynthesis de novo in plants. They reveal that in the absence of substrate, PDX1.3—in contrast to its bacterial counterparts—adopts a catalytically poised conformation, in which several structural features around the P1 active site are in place. A water-filled cavity is 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 presence of the pentose phosphate substrate, an intermediate corresponding to the predicted chromophoric adduct molecule at the P1 site is observed, and the mouth of the cavity is closed off by reorientation of a glutamate residue. In this PDX1.3-adduct structure, the catalytic aspartate, D40, is in an attacking conformation, extended toward the adduct at the center of the P1 site. Significantly, K165 previously thought to coordinate the P1 and P2 active sites has pivoted from its resting position in the P2 site to the P1 site and provides a clear demonstration of a swinging arm mechanism. A lysine-mediated swinging arm mechanism has been described previously for a variety of different protein families, including Class I aldolases (21), 2-oxoacid dehydrogenases, and the H protein of glycine decarboxylases (reviewed in ref. 28). However, they differ from that described here in that they either occur within a single active center (class I aldolases) or mediate shuttling of coenzyme bound intermediates. PDX1 makes use of the lysine swinging arm to coordinate distal divergent reaction sites, facilitating enzyme chemistry that notably has no need for a coenzyme intermediate. This thus provides an elegant example of how a sequence of biochemical Robinson et al.
reactions can be conducted by a single enzyme with no need to recruit distinct partners or remodel an active site. In summary, we have captured key snapshots of the plant PDX1 in action, helping to define the intricate workings of a highly complicated enzyme, essential for plant survival and important as a source of vitamin B6 to animals including humans. Methods Protein Expression and Purification. The construct pET–PDX1.3 containing the PDX1 homolog from Arabidopsis designed for expression of the protein with 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 1 mM IPTG, followed by growth for 6 h at 28 °C (30). For protein purification, the cell pellet from 1 L of expression culture was resuspended in 100 mM sodium phosphate buffer, pH 7.5, containing 300 mM NaCl, 10 mM imidazole, 1 mM β-mercaptoethanol, and 1 mM PMSF (Buffer A). Lysozyme (0.3 mg·mL−1), DNaseI (50 μg·mL−1), and a protease inhibitor mixture (EDTA-free; Roche) were added to the cell suspension, and lysis was performed by sonication. Insoluble material was removed by centrifugation (18,000 × g for 30 min at 4 °C), and the clarified lysate was applied to a gravity flow column containing 0.5 mL nickel-nitrilotriacetic acid affinity resin (Macherey–Nagel) 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 by 25 mL of Buffer A containing 50 mM imidazole. Protein was eluted from the column with Buffer A containing 300 mM imidazole and concentrated to 500 μL with partial buffer exchange (1:1) to 20 mM Tris·HCl, pH 7.0, containing 200 mM potassium chloride and 10 mM DTT followed by application to a preequilibrated Superdex200 (10/300; GE Healthcare) at 0.4 mL·min−1. Fractions containing the dodecameric form of PDX1.3 (30) were collected
PNAS | Published online September 19, 2016 | E5827
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Fig. 6. Conformational stability of the PDX1.3-adduct structure parallels a catch and pin. (A–C) Three features define conformational stability in the Arabidopsis PDX1.3-adduct (beige) upon comparison with its bacterial counterpart from G. stearothermophilus (cyan and gray). A proline residue, P152, in the 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 β6 and α6 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 PDX1.3adduct structures (Right). An increase in thermal motion is indicated by a shift in color from blue to red.
A 2
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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 interaction, 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 infrared absorbance using the Direct Detect system (Merck Millipore). Crystallization and Data Collection. Crystals were grown at 18 °C by sittingdrop vapor diffusion. Crystallization drops, consisting of 5 μL protein solution (2 mg·mL−1) and 5 μL precipitant solution [100 mM Mes·OH, pH 6.5, containing 1.4 M ammonium sulfate and 10% (vol/vol) 1,4-dioxane], were equilibrated against a 1-mL reservoir of precipitant solution. Crystals with rhombohedral morphology (space group H3; 146) and unit cell dimensions 178.5 Å × 178.5 Å × 115.8 Å appeared after 3–5 d and reached maximum dimensions (100 μm × 50 μm × 50 μm) after 4 wk. Crystals were recovered with nylon loops (Molecular Dimensions) and transferred to crystallization solution supplemented with 5% (vol/vol) ethylene glycol. This solution was
E5828 | www.pnas.org/cgi/doi/10.1073/pnas.1608125113
replaced in a stepwise manner to obtain a final solution containing 20% (vol/vol) ethylene glycol in 5% increments (3 min per step). Cryoprotected crystals were plunge-frozen in liquid nitrogen for storage and X-ray diffraction experiments. Crystals containing the chromophoric adduct were obtained by preparing crystals of apo-PDX1.3 as above, but cryoprotection solutions were supplemented with 6 mM R5P, and crystals were equilibrated for time intervals of 10 min to 27 h. A total of 22 diffraction datasets were obtained from PXI at the Swiss Light Source (PSI) at a wavelength of 0.9786 nm. Crystals of apo-PDX1.3 and PDX1.3-adduct diffracted to 1.46 Å and 1.76 Å, respectively. For data collection statistics see Table S1. PDX1.3 Chromophoric Intermediate Formation. To mimic formation of the previously described chromophoric intermediate in PDX1 (10, 38), formation
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Structure Solution and Refinement. Diffraction data were processed with programs of the CCP4 suite (39). Diffraction images were analyzed and integrated with MOSFLM (40, 41), and structure factors were obtained with SCALA (42). The PDX1.3 structure was determined by molecular replacement using the program PHASER MR (43) using a model prepared with CHAINSAW (44) based on the B. subtilis PDX1 homolog (2NV2). Phases for the chromophoric intermediate were obtained using the apostructure as a search model. Model building and refinement were performed with COOT (45) and REFMAC5 (46), respectively, and coordinates and chemical restraints for the
1. Eliot AC, Kirsch JF (2004) Pyridoxal phosphate enzymes: Mechanistic, structural, and evolutionary considerations. Annu Rev Biochem 73:383–415. 2. Müller S, Kappes B (2007) Vitamin and cofactor biosynthesis pathways in Plasmodium and other apicomplexan parasites. Trends Parasitol 23(3):112–121. 3. Fitzpatrick TB, et al. (2007) Two independent routes of de novo vitamin B6 biosynthesis: Not that different after all. Biochem J 407(1):1–13. 4. Burns KE, Xiang Y, Kinsland CL, McLafferty FW, Begley TP (2005) Reconstitution and biochemical characterization of a new pyridoxal-5′-phosphate biosynthetic pathway. J Am Chem Soc 127(11):3682–3683. 5. Raschle T, Amrhein N, Fitzpatrick TB (2005) On the two components of pyridoxal 5′-phosphate synthase from Bacillus subtilis. J Biol Chem 280(37):32291–32300. 6. Belitsky BR (2004) Physical and enzymological interaction of Bacillus subtilis proteins required for de novo pyridoxal 5′-phosphate biosynthesis. J Bacteriol 186(4):1191–1196. 7. Hanes JW, et al. (2008) Mechanistic studies on pyridoxal phosphate synthase: The reaction pathway leading to a chromophoric intermediate. J Am Chem Soc 130(10): 3043–3052. 8. Hanes JW, Keresztes I, Begley TP (2008) 13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase. Nat Chem Biol 4(7):425–430. 9. Hanes JW, Keresztes I, Begley TP (2008) Trapping of a chromophoric intermediate in the Pdx1-catalyzed biosynthesis of pyridoxal 5′-phosphate. Angew Chem Int Ed Engl 47(11):2102–2105. 10. Raschle T, et al. (2007) Reaction mechanism of pyridoxal 5′-phosphate synthase. Detection of an enzyme-bound chromophoric intermediate. J Biol Chem 282(9):6098–6105. 11. Guédez G, et al. (2012) Assembly of the eukaryotic PLP-synthase complex from Plasmodium and activation of the Pdx1 enzyme. Structure 20(1):172–184. 12. Moccand C, Kaufmann M, Fitzpatrick TB (2011) It takes two to tango: Defining an essential second active site in pyridoxal 5′-phosphate synthase. PLoS One 6(1):e16042. 13. Neuwirth M, et al. (2009) X-ray crystal structure of Saccharomyces cerevisiae Pdx1 provides insights into the oligomeric nature of PLP synthases. FEBS Lett 583(13): 2179–2186. 14. Raschle T, et al. (2009) Intersubunit cross-talk in pyridoxal 5′-phosphate synthase, coordinated by the C terminus of the synthase subunit. J Biol Chem 284(12):7706–7718. 15. Smith AM, Brown WC, Harms E, Smith JL (2015) Crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate (PLP) synthase. J Biol Chem 290(9):5226–5239. 16. Strohmeier M, et al. (2006) Structure of a bacterial pyridoxal 5′-phosphate synthase complex. Proc Natl Acad Sci USA 103(51):19284–19289. 17. Zein F, et al. (2006) Structural insights into the mechanism of the PLP synthase holoenzyme from Thermotoga maritima. Biochemistry 45(49):14609–14620. 18. Zhang X, et al. (2010) Structural insights into the catalytic mechanism of the yeast pyridoxal 5-phosphate synthase Snz1. Biochem J 432(3):445–450. 19. Dunn MF, Niks D, Ngo H, Barends TR, Schlichting I (2008) Tryptophan synthase: The workings of a channeling nanomachine. Trends Biochem Sci 33(6):254–264. 20. Mouilleron S, Golinelli-Pimpaneau B (2007) Conformational changes in ammoniachanneling glutamine amidotransferases. Curr Opin Struct Biol 17(6):653–664. 21. Du J, Say RF, Lü W, Fuchs G, Einsle O (2011) Active-site remodelling in the bifunctional fructose-1,6-bisphosphate aldolase/phosphatase. Nature 478(7370):534–537. 22. Hilario E, et al. (2016) Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water. Biochim Biophys Acta 1864(3):268–279. 23. Reed LJ (1998) From lipoic acid to multi-enzyme complexes. Protein Sci 7(1):220–224. 24. Knowles JR (1989) The mechanism of biotin-dependent enzymes. Annu Rev Biochem 58:195–221. 25. Gipson P, et al. (2010) Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy. Proc Natl Acad Sci USA 107(20):9164–9169.
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PNAS PLUS
chromophoric intermediate were generated with JLIGAND (47). Validation was performed with COOT (45), PDB_REDO (48), and MOLPROBITY (49), and structural analysis was performed with PYMOL and BAVERAGE (39). Refinement statistics are presented in Table S1. The resolvable region of the apostructure from Arabidopsis (residues 21–296, chain A) was aligned with structures from S. cerevisiae (3O07, Chain A), B. subtilis (2NV2, Chain A), and G. stearothermophilus (4WXY, Chain A) (15, 16, 18). Alignment was performed with SUPER in PYMOL using Cα atoms and with no cycles of refinement (50). Figs. 2–7 were prepared with PYMOL (50), and density maps in Figs. 2 and 4 were prepared with PHENIX (51). ACKNOWLEDGMENTS. We thank Vincent Olieric, Tomizaki Takashi, and all staff at the Swiss Light Source for assistance, as well as Stéphane Thore (University of Bordeaux) for critical analysis of structural refinements. Financial support is gratefully acknowledged from the Swiss National Science Foundation (Grant 31003A-141117/1 to T.B.F.) as well as the University of Geneva.
26. Leibundgut M, Jenni S, Frick C, Ban N (2007) Structural basis for substrate delivery by acyl carrier protein in the yeast fatty acid synthase. Science 316(5822):288–290. 27. Perham RN, Reche PA (1998) Swinging arms in multifunctional enzymes and the specificity of post-translational modification. Biochem Soc Trans 26(3):299–303. 28. Perham RN (2000) Swinging arms and swinging domains in multifunctional enzymes: Catalytic machines for multistep reactions. Annu Rev Biochem 69:961–1004. 29. Zhu J, Burgner JW, Harms E, Belitsky BR, Smith JL (2005) A new arrangement of (beta/ alpha)8 barrels in the synthase subunit of PLP synthase. J Biol Chem 280(30): 27914–27923. 30. Moccand C, et al. (2014) The pseudoenzyme PDX1.2 boosts vitamin B6 biosynthesis under heat and oxidative stress in Arabidopsis. J Biol Chem 289(12):8203–8216. 31. Kim S, Kim KJ (2013) Crystal structure of Mycobacterium tuberculosis Rv2606c: A pyridoxal biosynthesis lyase. Biochem Biophys Res Commun 435(2):255–259. 32. Matsuura A, Yoon JY, Yoon HJ, Lee HH, Suh SW (2012) Crystal structure of pyridoxal biosynthesis lyase PdxS from Pyrococcus horikoshii. Mol Cells 34(4):407–412. 33. Ramachandran GN, Sasisekharan V (1968) Conformation of polypeptides and proteins. Adv Protein Chem 23:283–438. 34. Newby ZER, et al. (2008) Crystal structure of the aquaglyceroporin PfAQP from the malarial parasite Plasmodium falciparum. Nat Struct Mol Biol 15(6):619–625. 35. Abramson J, et al. (2003) Structure and mechanism of the lactose permease of Escherichia coli. Science 301(5633):610–615. 36. Kumar H, Finer-Moore JS, Kaback HR, Stroud RM (2015) Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site. Proc Natl Acad Sci USA 112(29):9004–9009. 37. Tambasco-Studart M, et al. (2005) Vitamin B6 biosynthesis in higher plants. Proc Natl Acad Sci USA 102(38):13687–13692. 38. Tambasco-Studart M, Tews I, Amrhein N, Fitzpatrick TB (2007) Functional analysis of PDX2 from Arabidopsis, a glutaminase involved in vitamin B6 biosynthesis. Plant Physiol 144(2):915–925. 39. Winn MD, et al. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67(Pt 4):235–242. 40. Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG (2011) iMOSFLM: A new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67(Pt 4):271–281. 41. Leslie AG (2006) The integration of macromolecular diffraction data. Acta Crystallogr D Biol Crystallogr 62(Pt 1):48–57. 42. Evans PR (2011) An introduction to data reduction: Space-group determination, scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr 67(Pt 4):282–292. 43. McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Cryst 40(Pt 4):658–674. 44. Stein N (2008) CHAINSAW: A program for mutating pdb files used as templates in molecular replacement. J Appl Cryst 41(3):641–643. 45. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501. 46. Murshudov GN, et al. (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr 67(Pt 4):355–367. 47. Lebedev AA, et al. (2012) JLigand: A graphical tool for the CCP4 template-restraint library. Acta Crystallogr D Biol Crystallogr 68(Pt 4):431–440. 48. Joosten RP, Long F, Murshudov GN, Perrakis A (2014) The PDB_REDO server for macromolecular structure model optimization. IUCrJ 1(Pt 4):213–220. 49. Chen VB, et al. (2010) MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66(Pt 1):12–21. 50. DeLano WL (2002) The PyMOL Molecular Graphics System (Schrodinger LLC, San Carlos, CA). 51. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221.
PNAS | Published online September 19, 2016 | E5829
BIOCHEMISTRY
of the adduct was assessed in solution under cryoprotection conditions using a 96-well microplate reader (Synergy2; BioTek). The stoichiometry of the chromophore relative to total protein was estimated from the absorbance maxima at 282 nm and 310 nm for PDX1.3 and chromophore, respectively, and the respective extinction coefficients of 5,960 M−1·cm−1 (estimated from the PDX1.3 primary sequence) and 16,200 M−1·cm−1 (10).
