vitamin b6 biosynthesis: charting the mechanistic landscape

9
DOI: 10.1002/cbic.201000084 Vitamin B 6 Biosynthesis: Charting the Mechanistic Landscape Teresa B. Fitzpatrick,* Cyril Moccand, and CȖline Roux [a] This paper is dedicated to Nikolas Amrhein (ETH Zɒrich) for his outstanding support over the years together in Zɒrich unravelling the alternative route of vitamin B 6 biosynthesis in micro-organisms and plants. 1. Introduction Vitamin B 6 was discovered almost a century ago and is well es- tablished as an indispensable cofactor for the action of many enzymes, predominantly those involved in amino acid metabo- lism. The biosynthesis of the vitamin was extensively studied in Escherichia coli during the 1980s and 1990s. [1] However, the dis- covery of novel genes involved in tolerance to reactive oxygen species in fungi between 1998 and 2002 serendipitously impli- cated a completely novel pathway to the synthesis of the vita- min in the majority of organisms, distinct from that in E. coli. [2] This chance discovery led to a flurry of research activity in the ensuing years by several groups in attempts to unravel the nature of the alternative pathway. The importance of the vita- min is emphasized by the fact that it is an essential compound for all organisms, not only as a cofactor but also in its recently discovered role as an antioxidant; [2a, 3] only microorganisms and plants can make it (the majority employing the most recently discovered route), while higher organisms must take it in their diet. Therefore, the de novo biosynthesis of the vitamin by pathogenic or debilitating organisms represents a potential drug target and thus, the details of the mechanism of its syn- thesis are important for a rational approach to drug discovery. This review will summarize and reflect on the recent efforts to unravel the mechanism of vitamin B 6 biosynthesis with a par- ticular emphasis on the most commonly utilized of the two routes. 2. Two Routes to de novo Vitamin B 6 Biosynthesis The two routes to vitamin B 6 biosynthesis can be distinguished by the nature of the substrates used (Scheme 1). The less pop- ular route employed by E. coli and a few other members of the g-division of proteobacteria, utilizes deoxyxylulose 5-phos- phate (DXP) and 4-phosphohydroxy-l-threonine as substrates in a condensation reaction resulting in the formation of pyri- doxine 5-phosphate (PNP) [1a, d] and can be referred to as DXP- dependent. [4] Six enzymes are required to get to PNP through this route, (namely GapA, PdxB, PdxF, DXP synthase, PdxA and PdxJ) with an additional enzyme needed (PdxH) to convert PNP to the cofactor form, pyridoxal 5-phosphate (PLP). [5] On the other hand, most other micro-organisms, including arch- aea, fungi and the majority of eubacteria, in addition to plants, utilize ribose 5-phosphate (R5P) and glyceraldehyde 3-phos- phate (G3P) and just two enzymes Pdx1 and Pdx2 to directly synthesize the cofactor form, PLP. [2a, 6] As the latter pathway does not utilize DXP, it is referred to as DXP-independent. [4] Indeed, while the DXP-independent pathway would appear to be an elegant and rather simple route to the synthesis of PLP, the unravelling of its mechanism, the work of which is pre- dominantly undertaken by one enzyme Pdx1 (see below), is far from trivial. The two enzymes involved in the DXP-independ- ent route, Pdx1 and Pdx2, act together as a glutamine amido- transferase; Pdx2 acts as the glutaminase hydrolyzing gluta- mine, the ammonia product of which is passed onto Pdx1. [6c, 7] The latter on its own, through a series of yet to be fully deci- phered reactions synthesizes PLP utilizing R5P, G3P and ammo- nia. [6a, c] As a comparison of the DXP-dependent and -independ- ent routes to PLP has recently been covered, [5] here the focus will be on the emerging mechanistic details of the remarkable Pdx1:Pdx2 glutamine amidotransferase complex now known as PLP synthase. 2.1 Disparity in the oligomeric state of PLP synthase The Pdx1 and Pdx2 subunits of PLP synthase are separately synthesized and according to the X-ray crystallographic struc- ture of the bacterial complex from either Bacillus subtilis or Thermotoga maritima assemble as a 24-mer complex consisting of 12 Pdx1 subunits arranged as two opposing hexameric rings to which 12 individual Pdx2 subunits attach (Figure 1 A). [8] That this architecture exists in solution is supported by analytical ul- tracentrifugation studies, which demonstrated that Pdx1 alone exists in a hexamer-dodecamer equilibrium (K D = 3.7 mm), while Pdx1 within the assembled Pdx1-Pdx2 complex is solely a do- decamer to which twelve Pdx2 units attach. [8a] Each individual Pdx1 protomer assembles as a (b/a) 8 barrel with the dodeca- meric assembly being held in place through the elongation of helix a6 and the insertion of two additional helices a6and a6’’, the latter deviating from the canonical (b/a) 8 barrel. Inter- estingly, the Pdx1 enzyme from Saccharomyces cerevisiae has recently been shown to exist exclusively as a hexamer both in [a] Prof. T. B. Fitzpatrick, C. Moccand, Dr. C. Roux Department of Botany and Plant Biology, University of Geneva Sciences III, 30 Quai Ernest Ansermet, 1211 Geneva 4 (Switzerland) E-mail : [email protected] ChemBioChem 2010, 11, 1185 – 1193 # 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1185

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Page 1: Vitamin B6 Biosynthesis: Charting the Mechanistic Landscape

DOI: 10.1002/cbic.201000084

Vitamin B6 Biosynthesis: Charting the MechanisticLandscapeTeresa B. Fitzpatrick,* Cyril Moccand, and C�line Roux[a]

This paper is dedicated to Nikolas Amrhein (ETH Z�rich) for his outstanding support over the years together in Z�rich unravellingthe alternative route of vitamin B6 biosynthesis in micro-organisms and plants.

1. Introduction

Vitamin B6 was discovered almost a century ago and is well es-tablished as an indispensable cofactor for the action of manyenzymes, predominantly those involved in amino acid metabo-lism. The biosynthesis of the vitamin was extensively studied inEscherichia coli during the 1980s and 1990s.[1] However, the dis-covery of novel genes involved in tolerance to reactive oxygenspecies in fungi between 1998 and 2002 serendipitously impli-cated a completely novel pathway to the synthesis of the vita-min in the majority of organisms, distinct from that in E. coli.[2]

This chance discovery led to a flurry of research activity in theensuing years by several groups in attempts to unravel thenature of the alternative pathway. The importance of the vita-min is emphasized by the fact that it is an essential compoundfor all organisms, not only as a cofactor but also in its recentlydiscovered role as an antioxidant;[2a, 3] only microorganisms andplants can make it (the majority employing the most recentlydiscovered route), while higher organisms must take it in theirdiet. Therefore, the de novo biosynthesis of the vitamin bypathogenic or debilitating organisms represents a potentialdrug target and thus, the details of the mechanism of its syn-thesis are important for a rational approach to drug discovery.This review will summarize and reflect on the recent efforts tounravel the mechanism of vitamin B6 biosynthesis with a par-ticular emphasis on the most commonly utilized of the tworoutes.

2. Two Routes to de novo Vitamin B6Biosynthesis

The two routes to vitamin B6 biosynthesis can be distinguishedby the nature of the substrates used (Scheme 1). The less pop-ular route employed by E. coli and a few other members of theg-division of proteobacteria, utilizes deoxyxylulose 5-phos-phate (DXP) and 4-phosphohydroxy-l-threonine as substratesin a condensation reaction resulting in the formation of pyri-doxine 5’-phosphate (PNP)[1a, d] and can be referred to as DXP-dependent.[4] Six enzymes are required to get to PNP throughthis route, (namely GapA, PdxB, PdxF, DXP synthase, PdxA andPdxJ) with an additional enzyme needed (PdxH) to convertPNP to the cofactor form, pyridoxal 5’-phosphate (PLP).[5] Onthe other hand, most other micro-organisms, including arch-aea, fungi and the majority of eubacteria, in addition to plants,utilize ribose 5-phosphate (R5P) and glyceraldehyde 3-phos-

phate (G3P) and just two enzymes Pdx1 and Pdx2 to directlysynthesize the cofactor form, PLP.[2a, 6] As the latter pathwaydoes not utilize DXP, it is referred to as DXP-independent.[4]

Indeed, while the DXP-independent pathway would appear tobe an elegant and rather simple route to the synthesis of PLP,the unravelling of its mechanism, the work of which is pre-dominantly undertaken by one enzyme Pdx1 (see below), is farfrom trivial. The two enzymes involved in the DXP-independ-ent route, Pdx1 and Pdx2, act together as a glutamine amido-transferase; Pdx2 acts as the glutaminase hydrolyzing gluta-mine, the ammonia product of which is passed onto Pdx1.[6c, 7]

The latter on its own, through a series of yet to be fully deci-phered reactions synthesizes PLP utilizing R5P, G3P and ammo-nia.[6a, c] As a comparison of the DXP-dependent and -independ-ent routes to PLP has recently been covered,[5] here the focuswill be on the emerging mechanistic details of the remarkablePdx1:Pdx2 glutamine amidotransferase complex now known asPLP synthase.

2.1 Disparity in the oligomeric state of PLP synthase

The Pdx1 and Pdx2 subunits of PLP synthase are separatelysynthesized and according to the X-ray crystallographic struc-ture of the bacterial complex from either Bacillus subtilis orThermotoga maritima assemble as a 24-mer complex consistingof 12 Pdx1 subunits arranged as two opposing hexameric ringsto which 12 individual Pdx2 subunits attach (Figure 1 A).[8] Thatthis architecture exists in solution is supported by analytical ul-tracentrifugation studies, which demonstrated that Pdx1 aloneexists in a hexamer-dodecamer equilibrium (KD = 3.7 mm), whilePdx1 within the assembled Pdx1-Pdx2 complex is solely a do-decamer to which twelve Pdx2 units attach.[8a] Each individualPdx1 protomer assembles as a (b/a)8 barrel with the dodeca-meric assembly being held in place through the elongation ofhelix a6 and the insertion of two additional helices a6’ anda6’’, the latter deviating from the canonical (b/a)8 barrel. Inter-estingly, the Pdx1 enzyme from Saccharomyces cerevisiae hasrecently been shown to exist exclusively as a hexamer both in

[a] Prof. T. B. Fitzpatrick, C. Moccand, Dr. C. RouxDepartment of Botany and Plant Biology, University of GenevaSciences III, 30 Quai Ernest Ansermet, 1211 Geneva 4 (Switzerland)E-mail : [email protected]

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Scheme 1. The boxed area depicts the routes to the biosynthesis of vitamin B6 through the DXP-dependent, DXP-independent and salvage pathways. On theright is shown the internal aldimine that occurs between pyridoxal 5’-phosphate (PLP) and a lysine residue in a PLP-dependent enzyme, which upon entry ofthe substrate amino acid is converted to an external aldimine.

Figure 1. Structure of A) the Bacillus subtilis Pdx1/Pdx2 complex (PDB ID: 2NV2) and B) a single protomer, with the Pdx1 subunit depicted in green and thePdx2 subunit in grey. C) Superposition of the Gly46/Gly47 peptide from the B. subtilis Pdx2 structure (in blue, PDB ID: 2NV0) with the one from the glutaminebound dodecameric complex (in orange, PDB ID: 2NV2). Glutamine is depicted in pink. D) Superposition of the Gly46/Gly47 (B. subtilis numbering) peptidefrom the Thermotoga maritima glutamine free complex (in yellow, PDB ID: 2ISS) with the one from the glutamine bound dodecameric complex (in orange,PDB ID: 2NV2).

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solution and from X-ray crystallographic determination stud-ies.[9] There are three homologues of Pdx1 in S. cerevisiae(ScPdx1.1, ScPdx1.2, ScPdx1.3) and each one has a small se-quence insertion in the a6–a6’’ region not found in otherPdx1 homologues. It appears that these additional amino acidsare responsible for upholding the hexameric state in yeast, akey player being a single lysine residue (K177 in ScPdx1.1), asengineering of this extra residue into the B. subtilis enzymeresulted in the preferential formation of hexamers.[9] The dispa-rate observation of either hexamer or dodecamer in a particu-lar organism begs the question as to whether it has physiologi-cal implications. For example, S. cerevisiae PLP synthase hasbeen reported to have a catalytic activity almost fourfoldhigher than that of its bacterial or plant homologues.[9] There-fore, product output exceeds that of its orthologues and couldrepresent the evolution of a remarkable single site amino acidinsertion, that is, K177, resulting in a hexamer that surpassesthe efficiency of its dodecameric counterparts. In addition,there are regions observed in the X-ray structure of the auton-omous ScPdx1.1 protein that are not resolved in the structuresof the autonomous bacterial counterparts. One of these re-gions, helix a2’ (between b2 and a2, Figure 2) partially caps

the R5P binding site in Pdx1 and while it has been implicatedin enzyme activation, the exact biochemical nature of its func-tion has yet to be elucidated. In the determined bacterialstructures, this domain is only observed in the assembledPdx1:Pdx2 complex and upon R5P binding moves to tightenthe Pdx1 active site suggesting a role in catalysis.[5] It is note-worthy that the assembly of the bacterial complex not onlydiffers from autonomous Pdx1 by the formation of an extra

helix a2’, but also by an ordering of the N terminus to formhelix, aN, as well as strand bN25. Twenty-four amino acids atthe C-terminal region could not be resolved in the B. subtilisstructure.[8a] However, the X-ray structure of the enzyme fromT. maritima with R5P bound revealed ten residues extra,[8b]

while the apoenzyme from yeast resolved even further by anadditional 4 residues.[9] Yet, the remainder of the C terminus(ca. 10–15 residues depending on the source) has so fareluded structural characterization. A very recent study from usdemonstrated that indeed the C-terminal region of Pdx1 ishighly flexible and it has an essential role to play in catalysis.[10]

Moreover, crosslinking studies employing an unnatural aminoacid indicated that it manifests its role by acting as a molecularlever reaching over to an adjacent protomer, most likely withinone hexameric unit and assists in capping the adjacent activesite promoting catalysis. Indeed, the implicit dynamics as wellas the ornate architecture of PLP synthase in addition to itsmultiple catalytic abilities (see below) suggests regulation onseveral levels.

2.2 Transient observations within the PLP synthase complex

In the PLP synthase complex, the glutaminase and PLP synthe-sis active sites are remote from each other,[8] thus there aretwo disparate active sites that must communicate with eachother to ensure optimum activity and coordination of the syn-thesis of the PLP molecule. In this context, PLP synthase hasbecome another member of the substrate channelling machi-neries and serves as a model (yet to be explored) for ammoniachannelling. A proposal for a methionine-enriched channel hasbeen put forward based on the observation of cavities insidethe b-barrel of Pdx1[8a] and has been partially validatedthrough site directed mutagenesis of the Arabidopsis pro-tein.[7d] While glutamine amidotransferases in general appearto vary in the mechanism and dynamics of ammonia tunnel-ling,[11] it is clear that in the case of PLP synthase the employ-ment of an ammonia channel is transient. The available struc-tures of the Pdx1:Pdx2 complex either in the presence of thePdx2 substrate glutamine or the Pdx1 substrate R5P do notdisplay a clear open route for ammonia transfer. Therefore, thenature of what triggers the opening (and closing) of this chan-nel remain to be established, insight into which could be pro-vided in the future by molecular dynamics simulations and bythe availability of a structure which displays the channel in itsopen form. It is worth mentioning that PLP synthase itself hasbeen proposed to be a transient enzyme complex. For exam-ple, we have previously surmised that under physiological con-ditions, Pdx2 might dissociate from Pdx1 following the deliveryof ammonia. In vitro experiments employing the glutamineanalogue (2S-5S)-2-amino-3-chloro-4,5-dihydro-5-isoazoleaceticacid (acivicin) in which we aimed to mimic the supposed thio-ester intermediate that occurs during glutamine hydrolysis andrepresents a state after ammonia delivery,[12] indicated thatPdx2 was no longer associated with Pdx1.[6c] Indeed, followingammonia delivery, it would be logical to assume that Pdx2 issurplus to requirements.

Figure 2. Structure of the Bacillus subtilis Pdx1/Pdx2 protomer with helicesand sheets annotated. The Pdx1 subunit is depicted in green and the Pdx2subunit in grey. The b-sheets are highlighted in lime green. The vicinity ofthe P1 and P2 sites and the binding site of the ribose 5-phosphate substrateare depicted in pink.

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2.3 The Pdx1:Pdx2 interface

Several reports have documented the molecular details of theinteraction of Pdx1 with Pdx2 and the necessity of this contactfor priming of glutaminase activity in Pdx2.[7c, 8] Pdx2 is a three-layered aba sandwich with Rossmann topology, the active siteof which is defined by the presence of the catalytic triad Cys-His-Glu, typical of type I glutaminases.[7c, 8, 13] The glutaminaseactivity of Pdx2 is partly facilitated through formation of anoxyanion hole in the active site that is orchestrated predomi-nantly by movements of the peptide region (amino acids 45–49 in B. subtilis Pdx2) between strand b3 and helix a3 (alsoknown as the oxyanion strand). In B. subtilis Pdx2, the move-ment changes the orientation of the peptide such that the ni-trogen of Gly46 points into, rather than away, from the activesite and in combination with Ala80 permits co-ordination ofthe transient negative charge that forms on the amide oxygenof glutamine during the catalytic cycle.[8a] The Pdx2 active siteis located at the interface with Pdx1 and through a comparisonof the structure of autonomous Pdx2, and Pdx2 in complexwith Pdx1, it could be demonstrated that the trigger for theoxyanion strand peptide switch occurs upon interaction ofPdx1 with Pdx2.[8a] In particular, the ordering of the N-terminalhelix, aN (Figure 2), of Pdx1 upon complex formation results ina reorientation of Gln10 in Pdx2 and loss of its hydrogen bondwith Gly47; instead a new one is formed with the peptide car-bonyl of Lys18 in Pdx1. This leads to the concomitant switch ofthe Gly46/47 peptide, such that the oxyanion hole is in place(Figure 1 C). Significantly, this switch does not appear todepend on the presence of glutamine as it is observed in thestructure of the Pdx1:Pdx2 binary complex both in the pres-ence and absence of glutamine[8] (Figure 1 D). This suggeststhat the reorganisation is necessary for glutamine binding aswell as hydrolysis and has led to the hypothesis that Pdx1 andPdx2 initially form a so-called “encounter” complex that istightened upon glutamine binding to form the Michaelis com-plex.[14] The latter has been demonstrated with the proteinsfrom B. subtilis as well as P. falciparum, where addition of gluta-mine tightens the Pdx1:Pdx2 interaction 23-fold and 29-fold,respectively, as measured by isothermal titration calorime-try.[14–15] Moreover, in the case of the proteins from P. falci-parum the binding constant for glutamine binding to Pdx2 isdecreased 280-fold in the presence of Pdx1 (7 mm and 25 mm,respectively) and would lend support to the Pdx1:Pdx2 en-counter complex.[15] However, it is noted that there were sub-stantial differences in the binding affinities observed for gluta-mine depending on the nature of the experiment. For exam-ple, in the case of P. falciparum, titration of Pdx1 to Pdx2 in thepresence of glutamine results in a binding affinity that is 357times tighter than that of the Pdx1:Pdx2 “encounter complex”titrated with glutamine (0.07 and 25 mm, respectively, based ondata in ref. [15]). This would appear to suggest that Pdx2loaded with glutamine could also be an encounter form forPdx1 and could favour complex formation. In this case, Pdx1would bind to Pdx2:glutamine upon which the aN helix ofPdx1 triggers the rearrangement of the Pdx2 active site poisingit for glutamine hydrolysis. Indeed, it must also be mentioned

that the active site of Pdx2 becomes covered by the aN helixof Pdx1 upon interaction and would therefore be inaccessibleto glutamine entry.[8] In addition, glutamine is present in milli-molar concentrations in the cell, therefore the KD of 7 mm forglutamine binding to Pdx2[15] would seem compatible with thisconcept. In either case, as the aN helix of Pdx1 caps the Pdx2glutaminase active site sequestering the glutamine substrate,it ensures seclusion of the released ammonia and moreoverprovides the opening to the channel that is used to transferthe ammonia to the Pdx1 active site, some 26 � away at theother end of the (b/a)8 barrel.[8a] The Pdx1 helix aN appears tobe locked in place through hydrophobic interactions describedin detail for the B. subtilis and P. falciparum proteins in ref. [15] ,and the ordering of the bN sheet at the very N terminus of theprotein that, as mentioned earlier, is only observed in the PLPsynthase complex. It remains to be resolved why this key N-terminal domain of Pdx1 is in fact the least conserved partwithin this protein family[7d] (Figure 3). However, a rathersimple comparison of this region allows the speculation thatwhile aN is necessary for glutaminase function as outlinedabove, bN mainly contacts a b-sheet at the side of Pdx2 (spe-cifically b7–b8, Figure 2) acting as a molecular clip. Therefore,it could be assumed that an extension of the N-terminal Pdx1b-sheet as could be envisaged for Pdx1 from P. falciparum(Figure 3) would only serve to further lock the Pdx1:Pdx2 com-plex in place and hence stabilize it. This observation has notgone without precedent as we have observed that pull-outand native-PAGE studies of P. falciparum Pdx1 and Pdx2, analo-gous to those described in refs. [6c] , [8a] , indicated a morestable PLP synthase complex than that observed with theB. subtilis proteins (T.B.F. , Lukas B�rkle, unpublished observa-tions). Indeed, a homology model of the P. falciparum PLP syn-thase complex suggested that the 2-amino acid N-terminal ex-tension would allow for up to four extra hydrogen-bond inter-actions.[15] However, the same study showed that while dele-tion of this 2-amino acid extension decreased the binding af-finity for Pdx2 in the presence of glutamine, it did not appearto be significant. Moreover, the latter study highlighted themore hydrophobic interaction interface present in the P. falci-parum Pdx1:Pdx2 binary complex and indeed the larger totalinterface area compared to that of its bacterial counterparts inthe absence of glutamine and would also likely explain thehigher propensity to observe the intact binary complex in pull-out and native-PAGE analyses as mentioned above.

From recent studies, it is apparent that in addition to the N-terminal region, an aspartate and a serine residue in Pdx1 (D99and S75 in B. subtilis, respectively) are important in the sub-strate-binding region of Pdx2 (T.B.F. , Thomas Raschle, unpub-lished observations and ref. [16]). In particular, according to theB. subtilis numbering, Ser75 in Pdx1 makes hydrogen-bondcontacts with Arg106 and Asn107 of Pdx2, as well as an indi-rect interaction to Glu48 through a water molecule; this causesa reorientation of these residues in the Pdx2 active site. Therole of Ser75 in Pdx1 appears to be exclusive to Pdx2, albeitweak. In cases in which Ser75 is mutated to an alanine, PLPsynthase activity is unaltered compared to wild-type (whenusing an ammonium salt as the nitrogen donor rather than

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Pdx2 and glutamine), while Pdx2 glutaminase activity is re-duced by 16 %.[16] On the other hand, Asp99 in Pdx1 pushesinto the Pdx2 active site and makes a salt bridge with Arg135of Pdx2. In so doing, it disrupts the salt bridge of the latterwith Asp114, while additionally causing an almost 908 reorien-tation of loop 7 in Pdx2 (i.e. , the region between b6 and b7 inPdx2, Figure 2). Replacement of Asp99 with an alanine resultsin an 83 % reduction in Pdx2 glutaminase activity and loss ofthe influence of glutamine on promoting a 1:1 stoichiometryof Pdx1 and Pdx2.[16] In this context, Asp99 could be likened toan “antenna” that senses and primes the Pdx2 active site.Moreover, its absolute conservation (in contrast to Ser75) high-lights its importance in co-ordination of Pdx2 activity. Notably,Glu48, Arg106 and Arg135 in Pdx2 are essential for glutami-nase activity, while Arg106 and Arg135 are also essential forthe interaction with Pdx1.[16]

3. The Pdx1 Active Site

As Pdx1 is mainly responsible for the chemical gymnastics nec-essary for the biosynthesis of the PLP molecule and with sucha complicated system in mind, it is no surprise that the use ofsite-directed mutagenesis has been used to complement thestructural analyses in order to pinpoint the role of individualamino acids and identify the catalytically important groups. Asis true of all (b/a)8 barrel proteins, the active site of Pdx1 wasexpected to be located at the top C-terminal end of thebarrel.[17] However, in the several Pdx1 structures determinedso far,[8, 18] sulfate, chloride or phosphate ions were seen tobind to two apparently specific sites, one of which is part ofthe expected active site and was later demonstrated to be theribulose 5-phosphate (Ru5P) binding site by the cocrystalliza-tion of PLP synthase with Ru5P bound[8b] and the second is lo-cated at the hexamer interface of the Pdx1 dodecamer.[8] The

Figure 3. Alignment of the deduced amino acid sequences of selected Pdx1 proteins. The asterisks and boxed residues represent amino acids that are catalyt-ic and/or involved in substrate binding in addition to a-helices and b-sheets, respectively. Where indicated the numbering used is for the Bacillus subtilis pro-tein (BsPdx1), with the exception of ScK177, which indicates the “extra residue” in the Saccharomyces cerevisiae protein. The sequences shown are from Arabi-dopsis thaliana (AtPDX1.1-AtPDX1.3), Proteus vulgaris (PvPdx1), Bacillus subtilis (BsPdx1), Staphylococcus aureus (SaPdx1), Thermotoga maritima (TmPdx1), Cer-cospora nicotianae (CnPdx1), Plasmodium falciparum (PfPdx1), Saccharomyces cerevisiae (ScPdx1.1-ScPdx1.3). The signature motif describes predicted phos-phate utilizing enzymes involved in pyridoxine/purine/histidine biosynthesis (COG database). The alignment was performed by using VectorNTI Advance 11.

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two separate sites are referred to as P1 (the site of R5P bind-ing) and P2 (Figure 2). The P1 site is made up from Asp24,Lys81, Asp102, Arg147, Gly153, Gly214, Gly235 and Ser236(B. subtilis numbering).[8] The amide nitrogen atoms of Gly153,Gly214, Gly235, Ser237 and the side chain of Ser237 were ob-served to form hydrogen bonds with the phosphate oxygenatoms of the substrate, while Arg147 and Asp102 were hydro-gen bonded to the C1 hydroxyl group, and Asp24 was hydro-gen bonded to the C3 hydroxyl group.[8b] While a catalytic rolehas been confirmed for Arg147, Asp102 and Asp24, the exactmolecular nature of this role requires definition, however theyare thought to be involved in proton shuffling[8a] (T.B.F. , C.M.,unpublished observations). In the crystal structure of PLP syn-thase, Lys81 was interpreted to be in an imine adduct forma-tion with Ru5P[8b] and furthermore, was thought to representone of the first steps in the catalytic mechanism of PLP syn-thase (see below). However, a second lysine residue (Lys149)that bridges the P1 and P2 active sites had originally been pro-posed to form this adduct with Ru5P.[6a] Indeed, the role of thissecond lysine residue has been a point of contention in the lit-erature,[5] but mass spectrometry and mutagenesis studies inaddition to the X-ray structure of PLP synthase with pentosephosphate substrate bound, as mentioned above, have lentlittle support to the original report with all subsequent studiesfavouring Lys81 as the residue responsible during this part ofthe reaction.[8b, 19] However, mutagenesis studies of Lys149 havedemonstrated that it does play a role in PLP synthesis and hasbeen suggested to act as a catalytic base at a later step duringthe course of the reaction.[19] This residue points towards theP2 site and therefore would imply that it is in this vicinity thatits function is executed. Although, it has been proposed thatLys149 could be repositioned by a “swinging” mechanism suchthat its NZ atom approaches the covalent adduct between R5Pand Lys81 in the P1 site.[8b] Given that in all structures exam-ined so far, Lys149 is somewhat “fixed” in its position with littleinherent side chain flexibility (as exemplified by the compara-tively low B factor value), the latter proposal seems unlikely.On the other hand it could be foreseen to play a role in thefunctionality of the P2 site. The latter site is made up fromHis115, Arg130, Glu134, Arg137 and Arg138, in addition toLys187 from a6’ from a neighbouring protomer on the adja-cent hexamer.[8, 18] This second site is mainly exposed and hasbeen suggested to represent the binding site of G3P, the prod-uct PLP or an intermediate along the reaction co-ordinate.[8b]

3.1 The catalytic mechanism of PLP synthase

Several mechanistic versions have been put forward so far,with each one advancing the proposals of the previous suchthat the evolving picture begins to attain clarity. Certain piecesof the puzzle have remained firmly in place. For example, it isaccepted that the reaction starts with the binding of the pen-tose phosphate substrate, R5P. It is thought that the furanosering of the latter is the form that the Pdx1 enzyme acceptsand the catalytic path commences with the opening of thering most likely followed by isomerization to Ru5P.[19] The nextstep is formation of a covalent adduct at an active site lysine

residue (Lys81 in B. subtilis, as discussed above).[8b, 19] At firstthis adduct was thought to be an imine with the C2 carbonylof Ru5P based on the fact that Pdx1 has R5P-Ru5P isomeraseactivity and is structurally similar to imidazole glycerol phos-phate synthase, which catalyses the formation of an imine atthe C2 carbonyl of a 1-aminoribulose 5-phosphate deriva-tive.[6a, 8b] More recently a series of elegant NMR experimentshave established that it is a C1�N bond between the sugarand the lysine residue, respectively.[20] Subsequently, loss ofwater and inorganic phosphate give rise to a highly conjugat-ed covalent adduct that is chromophoric as exemplified by itsabsorbance maximum at 315 nm.[19] Observation of this chro-mophoric intermediate marked a turning point in the relativelyshort history of the elucidation of the mechanism of PLP syn-thase, as it brought clarity to the sequence of the initial stepsof catalysis. The formation of the chromophoric intermediatewas shown to be absolutely dependent on the presence ofammonia either when added as an ammonium salt or as aresult of the glutaminase activity of Pdx2.[19] However, as ex-periments with 14N- and 15N-labelled ammonia did not revealthe presence of nitrogen in the chromophoric intermediateusing the mass spectrometric approach that initially docu-mented this intermediate, it was not included in the proposedstructure(s) at the time.[19] Later, a study in which the inter-mediate was dissociated from the enzyme in 7 m urea in thepresence of Tris(2-carboxyethyl)phosphine trapped a new spe-cies with a broad absorbance extending below 260 nm.[21] The1H NMR spectrum of the trapped species established that nitro-gen was incorporated. The latter provided an explanation as towhy formation of the chromophoric intermediate was depen-dent on an ammonia source.[19] It was deduced that the dis-crepancy in the original mass spectrometry study could beaccounted for if the suggested adduct underwent hydrolysisunder the acidic conditions used.[19, 21] As the rate of formationof the chromophoric intermediate (0.212 min�1) is substantiallyfaster than PLP formation (0.040 min�1), it is not the rate-limit-ing step of the overall reaction. However, a primary deuteriumisotope effect of 2.4 has been documented for removal of thepro-R proton at C5 of R5P and suggested that removal of thisproton is at least partially rate limiting in the formation of thechromophoric intermediate itself.[22] The ensuing removal ofphosphate occurs by elimination and exhibits the same kinet-ics as chromophore formation suggesting that both are limitedby the same rate constant(s), that is, removal of the C5 pro-Rproton. Furthermore, a C1 to C5 lysine migration has been pro-posed to occur during the chromophoric intermediate forma-tion and is a novel variation of imine chemistry of proteins.[20]

As pointed out in the latter study, such a shift would provide anew environment, enhance the catalytic versatility of theactive site and could perhaps account for the lack of obviousfunctional groups to mediate the chemistry behind this reac-tion based on the structure of R5P bound PLP synthase. Theformation of PLP from the chromophoric intermediate is com-pleted in vitro by simply adding G3P and in turn confirmedthe catalytic competency of the chromophore.[19, 21] The rate offormation of PLP from G3P and the chromophoric intermediate(0.030 min�1) approximates to the turnover of the entire reac-

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tion (0.040 min�1) and suggests that the overall rate-limitingstep occurs after G3P binding. However, the details of this partof the reaction remain to be explored and so far mechanismsinvolving a series of plausible steps to reach the final reactionproduct, PLP, have been proposed but not confirmed.[19–22] In-triguingly, the product of the reaction is difficult to separatefrom the protein (by dialysis, gel filtration, etc.) and indeed ithas recently been proposed that it is bound as an internal aldi-mine to the protein.[20] Therefore, an additional hydrolysis stepis necessary to release the product for incorporation into en-zymes that rely on it as a cofactor. It will be interesting to de-termine the nature of the trigger for this reaction. Envisionedpossibilities could include formation of an external aldiminethrough binding of an amino acid (which would incidentallyimply physiological sensing of the latter) or conformationalchanges mediated through interaction with an enzyme depen-dent on PLP, facilitating hydrolysis. Moreover, it will also be in-teresting to determine the product location at either the P1 orP2 site of Pdx1, which in turn will provide additional insightsinto the exact nature of the catalytic mechanism for the latterpart of the reaction, that is, after G3P binding. In either case,the fact that product release appears to be a rate-determiningstep additionally implies that Pdx1 represents a storage formfor PLP and prevents the release of a chemically instable alde-hyde into the cellular milieu; thus, this prevents fortuitous re-actions with other imines in the cell (that is, unspecific transa-mination reactions). Thus, while several molecular snapshotsalong the PLP synthase reaction coordinate have been cap-tured, we will refrain from suggesting yet another mechanisticproposal here until the latter steps of the reaction have beensolved.

4. Cooperativity and Allosteric Control of PLPBiosynthesis

Recently, both inter- and intrasubunit regulation has beendocumented for PLP synthase.[10] Specifically, subtle conforma-tional changes have begun to emerge for intra-subunit regula-tion in Pdx1, in particular at the C terminus. As this regioneluded X-ray structural characterisation[8, 18] and was shown tobe highly susceptible to partial protease cleavage,[10] it was as-sumed to have an inherent flexibility. Probing of this regionwas facilitated by the opportune location of a single trypto-phan residue at the very C terminus of the B. subtilis Pdx1enzyme and moreover, by it being the sole tryptophan residuein this source while its partner enzyme Pdx2 does not haveany such residues. Thus, fluorescence studies were used todemonstrate that Pdx1 could adopt several conformationalstates. The first state captured, which most likely representsthe resting state, is an open conformation ready to accept theprimary substrate R5P. Binding of the pentose phosphatesugar triggers another subtle conformational change in theC terminus, most likely related to the movement of a2’ anda8’ (Figure 2) in Pdx1 and thereby tightening the active site, ashas been demonstrated by a comparison of the crystal struc-ture of the protein complex with and without R5P present.[10]

Furthermore, the binding of R5P relays cooperativity through

the Pdx1 oligomer (Hill coefficient of about 3) implying intra-domain signalling. Interestingly, interaction with the catalytical-ly primed Pdx2 harbouring glutamine in a Michaelis complexcaptured yet another conformational state and substantiallyenhanced the affinity for the pentose phosphate substrate inPdx1 (about fourfold) and furthermore, demonstrates intersu-bunit allostery. Indeed, it could be shown in the same study,through stopped flow fluorescence spectroscopy, that bothPdx2 and glutamine contribute to this allosteric effect.[10] It willbe interesting to unravel the path of this relay from Pdx2,which dictates movement of the remote Pdx1 C terminus. Im-portantly, the C terminus was shown to be essential for catalyt-ic activity and its functionality appears to be mediated throughinteraction with a neighbouring protomer. Through the strate-gic incorporation of a photoactivatable unnatural amino acid(p-benzoyl-phenylalanine) followed by crosslinking studies andnanoliquid-chromatography-coupled mass spectrometry, it wassuggested that the C-terminal region of a single Pdx1 proto-mer acts like a lever and stretches over to a neighbouring pro-tomer. In doing so, it caps the adjacent active site facilitatingcatalysis.[10] Indeed, the combined cooperativity and intraproto-mer interaction might assist in explaining the ornate dodeca-meric architecture observed for Pdx1. The detailed structuralbasis of the effects on the C terminus, associated with an alter-ation in the equilibrium between alternative Pdx1 conforma-tions is still somewhat elusive, and might remain so until thestructure of the protein with the entire C terminus has been re-solved. In the aforementioned study, we reported that whenboth R5P and G3P are present, the C terminus is protectedfrom partial proteolytic digestion and thus might be necessaryin crystallization studies to observe complete ordering of theC terminus.

5. The Salvage Pathway of Vitamin B6Biosynthesis

While much has been overviewed so far about the de novobiosynthesis of vitamin B6, it must be kept in mind that thereis also a salvage pathway for this vitamin in place. Through thesalvage pathway the various vitamer forms (pyridoxine (PN),pyridoxal (PL), pyridoxamine (PM) and their corresponding 5’-phosphate esters) can be interconverted by the action of kin-ases, oxidases and transaminases[23] (Scheme 1). Through thisroute PN, PL or PM can be phosphorylated by the kinase,PdxK, to form PNP, PLP or PMP, respectively.[24] Interestingly,two kinases exhibiting different substrate specificities havebeen identified in E. coli : the aforementioned PdxK that cataly-ses conversion of all B6 vitamers to their respective 5’-phos-phate esters and PdxY that specifically uses PL as a substra-te.[1f] The salvage pathway also comprises the oxidation of PNPand PMP into PLP by an oxidase known as PdxH in bacteria[25]

and PDX3 in yeast[26] and plants.[27] It is only recently that thelatter enzyme has been characterised in plants.[27–28] Alterna-tively, PLP can be converted into PMP by transaminases.[29] Incontrast to the de novo pathway of vitamin B6 biosynthesisthat is restricted to microorganisms and plants, the salvagepathway is found in all organisms. However, by its very nature

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those organisms that cannot synthesize the vitamin de novomust take a form of the vitamin up with their diet.

6. Exploiting Vitamin B6 Biosynthesis as a DrugTarget

Compounds with potential as drugs should ideally be designedto target the organism accountable without harming the host.As vitamin B6 is essential for all organisms but de novo biosyn-thesis is restricted to microorganisms and plants and does nottake place in mammals, it would appear to fulfil the require-ments as a potential drug target. Indeed, organisms that cansynthesize the vitamin de novo include some well-knownpathogens and the pathway has been discussed several timesin the context of a putative drug target. Pathogenic organismsthat can catalyze its biosynthesis through the DXP-dependentpathway include Neisseria meningitidis, Salmonella typhimuri-um, Vibrio cholerae and Yersinia pestis.[30] The more recentlyidentified, but more predominant DXP-independent pathwayis present in most microorgan-isms (with the exception ofthose few that have the DXP-dependent pathway and certainobligate parasites that lackeither route[2a, d, 6b]) as well asplants.[4] Several pathogenic bac-teria employ the DXP-independ-ent pathway, for example, Myco-bacterium tuberculosis, Mycobac-terium leprae, Corynebacteriumdiphteriae in addition to the api-complexa. The apicomplexa in-clude such debilitating organ-isms as Toxoplasma gondii andP. falciparum. The latter is thecausative agent of the mostsevere form of malaria, well documented as one of the mostthreatening diseases in the world with more than 300 millionpeople infected and up to two million fatalities annually.[31] Theassumed essentiality of de novo vitamin B6 biosynthesis tothese organisms has led to discussion of inhibition of the path-way as a strategy for targeted drug development.[32] However,it remains to be established experimentally if de novo biosyn-thesis is indeed essential for the apicomplexa as well as thepathogenic bacteria mentioned, as they might also have thepotential to acquire the vitamin from their environment. Inter-estingly, disruption of the two functional PDX1 paralogues inArabidopsis thaliana, leads to an embryo-lethal phenotype.[4, 33]

A similar effect is observed by knocking out the single copyPDX2.[4] Therefore, a specific inhibitor of the PLP synthase com-plex in plants might have potential as an herbicide in agricul-ture. A substrate-based approach is complicated by the factthat the compounds used by the DXP-independent pathway,in particular, are not only abundant in the cell but are sub-strates for several essential metabolic enzymes (triose phos-phate isomerase, glyceraldehyde 3-phosphate dehydrogenase,ribose 5-phosphate isomerase, transketolase). On the other

hand, some of the unique features of PLP synthase describedabove such as helix aN in Pdx1, necessary for complex assem-bly and activity, or the required inherent flexibility of the Pdx1C terminus might provide strategies to rationally develop mol-ecules that will block assembly and activity, respectively.

Alternatively, many enzymes that are dependent on vita-min B6 as a cofactor and are essential for survival also offer thepossibility to target diseases.[34] Examples that have alreadybeen exploited include the inhibition of g-aminobutyric acid(GABA) aminotransferase with vigabatrin as an epilepsy thera-py, inhibition of alanine racemase as antibacterial agents andinhibition of ornithine decarboxylase as a target in cancer ther-apy as well as in the fight against African trypanosomes (thecausative agent of sleeping sickness).[35] Recently, a BOC-pro-tected pyridoxyl–ornithine conjugate that mimics the Schiffbase intermediate (Scheme 2) involved in enzymatic decarbox-ylation has been synthesized and has been shown to inhibitgrowth of various tumor cells more effectively than nontu-mourigenic cells.[36] Another mimic of the coenzyme–substrate

adduct that occurs during PLP catalysis, pyridoxyl-histidinemethyl ester, has been shown to inhibit human histidine decar-boxylase in human mastocytoma cells without affecting prolif-eration.[37] The PLP-dependent enzyme, ornithine decarboxy-lase, which is involved in polyamine formation and is essentialfor cellular proliferation and differentiation, has also been dis-cussed as an antimalarial target.[35] An approach to target theparasite was recently presented in which pyridoxyl-adductswere tested as antimalarials.[35] The strategy is to take advant-age of the presence of the PN/PL/PM kinase, PdxK, in P. falci-parum[7e] and proposes to feed the parasite with unphosphory-lated pyridoxyl-adducts, which then become phosphorylatedby PdxK and presumably get “trapped” within the parasite inthis state. As the compounds in question, pyridoxyl-tryptophanmethyl esters named PT3 and PT5, in their phosphorylatedstate mimic an intermediate along the normal PLP catalyticroute, that is, the external aldimine between PLP and trypto-phan, they are expected to bind to PLP-dependent enzymes,thereby causing their inhibition, preventing metabolism andkilling the parasite (Scheme 2). Indeed, the authors could showthat one of these compounds, PT3, has a respectable IC50 value

Scheme 2. Structures of the pyridoxyl-tryptophan external aldimine (PLP–Trp) and the corresponding methylesters PT3 and PT5.

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of 14 mm.[35] Moreover, overexpression of the PN/PL/PM kinase,PdxK, negatively affected the growth of the parasite in thepresence of PT3 compared to controls. However, this com-pound marginally affected the growth of mammalian cells(25 % decrease in growth at a concentration of 50 mm PT3) andit remains to be determined to what extent the parasite couldbe “rescued” by salvaging the vitamin from the erythrocyteduring human blood stage infection. Therefore, the propensityof vitamin B6 biosynthesis as a drug target will require knowl-edge not only of the biosynthesis but also of the mechanismsof uptake into the cell of the organism to be targeted.

Outlook

Thus, the overall emerging picture of vitamin B6 biosynthesisemphasizes the need to combine structural, dynamic and func-tional data. The multiple catalytic ability of Pdx1, the remoteglutaminase and synthase active sites and the elaborate struc-ture, disseminate regulation at several levels. Charting themechanistic landscape of PLP synthase has and will, withoutdoubt, involve much rough and challenging terrain with a mul-titude of unknown territories that will, most likely, provideobstinate remuneration to the several laboratories who haveresigned to tackle this demanding and chemically remarkableenzyme.

Acknowledgements

Financial support is gratefully acknowledged from the Swiss Na-tional Science Foundation (Grant PPOOA_119186) to T.B.F.

Keywords: (beta/alpha)8 barrel · ammonia tunnel · reactionmechanisms · vitamin B6 · vitamins

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Received: February 11, 2010Published online on April 15, 2010

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