Phosphate-Dependent Enzymes Involved in Biotin Biosynthesis: Structure, Reaction Mechanism and Inhibition☆

Biochimica et Biophysica Acta 1814 (2011) 1459–1466

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Review Pyridoxal-5′-phosphate-dependent enzymes involved in biotin biosynthesis: Structure, reaction mechanism and inhibition☆

Stéphane Mann, Olivier Ploux ⁎

Laboratoire Charles Friedel, ENSCP Chimie ParisTech, UMR CNRS 7223, 11 rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France article info abstract

Article history: The four last steps of biotin biosynthesis, starting from pimeloyl-CoA, are conserved among all the Received 5 July 2010 biotin-producing microorganisms. Two enzymes of this pathway, the 8-amino-7-oxononanoate synthase Received in revised form 4 November 2010 (AONS) and the 7,8-diaminopelargonic acid aminotransferase (DAPA AT) are dependent on pyridoxal-5′- Accepted 10 December 2010 phosphate (PLP). This review summarizes our current understanding of the structure, reaction mechanism Available online 21 December 2010 and inhibition on these two interesting enzymes. Mechanistic studies as well as the determination of the crystal structure of AONS have revealed a complex mechanism involving an acylation with inversion of Keywords: ﬁ ﬁ 8-amino-7-oxononanoate synthase con guration and a decarboxylation with retention of con guration. This reaction mechanism is shared by the 7,8-diaminopelargonic acid aminotransferase homologous 5-aminolevulinate synthase and serine palmitoyltransferase. While the reaction catalyzed by Pyridoxal-5′-phosphate DAPA AT is a classical PLP-dependent transamination, the inactivation of this enzyme by amiclenomycin, a Enzyme reaction mechanism natural antibiotic that is active against Mycobacterium tuberculosis, involves the irreversible formation of an Inhibition adduct between PLP and amiclenomycin. Mechanistic and structural studies allowed the complete description Amiclenomycin of this unique inactivation mechanism. Several potent inhibitors of these two PLP-dependent enzymes have been prepared and might be useful as starting points for the design of herbicides or antibiotics. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The ﬁrst steps of biotin biosynthesis, leading to pimeloyl-CoA, have not yet been completely unraveled and seem to be different in (+)-Biotin is a water-soluble vitamin (vitamin H) that is a cofactor for Escherichia coli [2] and Bacillus sphaericus [3], for instance. To the carboxylases, transcarboxylases and some decarboxylases. This molecule contrary, the four last steps, from pimeloy-CoA to biotin, as depicted is only biosynthesized by bacteria, plants and fungi but essential to all in Fig. 1, are all conserved in the biotin-producing organisms so far living organisms. The biosynthetic route to biotin was discovered in the studied. Among the four enzymes involved in this biosynthetic route, sixties and seventies and then studied by different groups [1].Lately, two are dependent on pyridoxal-5′-phosphate (PLP): the 8-amino-7- several scientiﬁc challenges such as the production of biotin by oxononanoate synthase (AONS) and the 7,8-diaminopelargonic acid fermentation, the design of new antibiotics or herbicides or just the aminotransferase (DAPA AT). This review summarizes our current fundamental study of unusual enzymatic reactions, led several laborato- knowledge on these two interesting enzymes. ries to launch new projects on biotin biosynthesis. This resulted in a number of original works, some of which are discussed in this review. 2. 8-Amino-7-oxononanoate synthase

The activity of this enzyme was first demonstrated in cell free Abbreviations: AdoMet, S-adenosyl-L-methionine; ALAS, 5-aminolevulinate extracts of E. coli by Eisenberg and Star [4] and later in a number of other synthase, EC 2.3.1.37; AON, 8-amino-7-oxononanoic acid (KAPA was also used in the microorganisms by Izumi et al. [5] who partially purified the enzyme older literature); AONS, 8-amino-7-oxononanoate synthase (KAPA synthase), EC from B. sphaericus [6]. However, it was only in 1992 that a pure 2.3.1.47; DAPA, 7,8-diaminononanoic acid; DAPA AT, 7,8-diaminopelargonic acid aminotransferase, EC 2.6.1.62; KBL, 2-amino-3-oxobutyrate CoA ligase, EC 2.3.1.29; preparation of the enzyme from B. sphaericus was reported [7] allowing KIE, kinetic isotope effect; PLP, pyridoxal-5′-phosphate; PMP, pyridoxamine-5′- subsequent detailed mechanistic studies on this enzyme [8]. Then, the phosphate; SPT, serine palmitoyltransferase, EC 2.3.1.50 enzymes from E. coli [9], Arabidopsis thaliana [10], Mycobacterium ☆ This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology. tuberculosis [11] and Thermus thermophilus [12],werepurified and ⁎ Corresponding author. Biochimie des micro-organismes, Laboratoire Charles Friedel, characterized. The enzyme from T. thermophilus has two activities, a ENSCP Chimie ParisTech, UMR CNRS 7223, 11 rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France. Tel.: +33 1 44 27 67 01; fax: +33 1 44 27 67 01. 2-amino-3-oxobutyrate CoA ligase (KBL) activity and a lower AONS E-mail address: [email protected] (O. Ploux). activity [12], but it has not yet been proved that this enzyme is actually

Fig. 1. The biosynthesis of biotin involves four steps starting from pimeloyl-CoA, two of which are catalyzed by PLP-dependent enzymes. involved in biotin biosynthesis. The enzymes from B. sphaericus and E. in the crevice formed by these domains (Fig. 2). This fold resembles coli were crystallized and the three-dimensional structure of the E. coli that of other PLP-dependent enzymes, such as the dialkylglycine enzyme was solved [13,14]. decarboxylase [15] and is very close to the fold of the three other AONS belongs to this interesting class of enzyme that catalyzes the α-oxoamine synthases [16–18] (Fig. 2), although the sequence condensation of an amino acid on a CoA-thioester with a concomitant identities within the α-oxoamine synthase family are weak. However, decarboxylation. Four enzymes, so-called α-oxoamine synthases, share the active site residues that are in direct contact with the PLP cofactor this reaction (Table 1), and it is now recognized that these synthases are and involved in catalysis are all conserved in the α-oxoamine synthase homologous with strong structural and mechanistic similarities. family, that is, His133, Ser179 Asp204, His207, Thr233, and Lys236 of the E. coli AONS (Fig. 3). 2.1. Steady-state kinetic parameters and molecular properties

The activity of AONS was first measured using a microbiological 2.3. AONS reaction mechanism assay [4–7], by detecting the 8-amino-7-oxononanoate (AON) product using Saccharomyces cerevisiae as the test organism, but The reaction mechanism of AONS was first studied by Ploux et al. more recently and less tediously, using the disappearance of the [8] on the B. sphaericus enzyme and they showed that it was similar to thioester chromophore [8] or either an enzymatic or colorimetric that proposed for 5-aminolevulinate synthase (ALAS) [19,20] and for detection of the CoASH product [9–11]. The kinetic parameters serine palmitoyltransferase (SPT) [21]. The central question was to obtained varied with the enzyme source (Table 2). It should be noted discriminate among the two plausible pathways: either the stabilized that the enzyme from M. tuberculosis showed a very low turnover carbanion was formed by decarboxylation and would then react on number, a rather intriguing and unexplained situation. The kinetic the thioester with retention of configuration, or it was formed by mechanism of AONS, a bisubstrate-biproduct enzyme, not counting proton abstraction followed by an acylation step and a decarboxyl- the consumed proton, has not been investigated and it is not known if ation involving one overall inversion of configuration. The latter it is random or ordered. However, L-alanine can form an external chemical mechanism was supported by several arguments. First, the 2 aldimine in the absence of pimeloyl-CoA [8] and the binding of C2-proton or C2-deuteron of L-alanine or L-[2- H]alanine was lost in pimeloyl-CoA in the absence of alanine has been observed [11], the reaction when these substrates were used in deuteriated water or pointing to a random mechanism. The enzymes from B. sphaericus [7] in water, respectively. Second, the B. spharericus AONS was able to and A. thaliana [10] behaved as monomers in solution but the E. coli catalyze a stereospecific exchange, with the solvent protons, with and B. sphaericus enzymes crystallized in a dimeric form, suggesting a retention of configuration of the C2-proton of L-alanine in the absence facile equilibrated dimerization. of the second substrate, as well as the exchange of the C8-proton of AON in the same conditions. Third, a deuterium kinetic isotope effect D 2.2. AONS three-dimensional structure (KIE), Vmax =1.3, compatible with a primary KIE, was observed using L-[2-2 H]alanine as the substrate, and a strong solvent KIE D2O AONS belongs to the PLP-dependent aminotransferase I (fold I) Vmax =4.0 was observed, compatible with a slow reprotonation superfamily and to the KBL like subfamily. The crystallographic step. Finally the enzyme catalyzed a slow abortive transamination in structures of the E. coli apo-, holo- and AON-bound enzyme forms the presence of L-alanine. Taken together all these experimental facts have been reported [9,14]. The structure of the B. sphaericus enzyme showed that AONS catalyzed the reaction using the mechanism was solved and it is very similar to that of the E. coli enzyme (O. Ploux, depicted in Fig. 3. Later on, using a phosphonate analog of the C. Cambillau, unpublished results). The E. coli enzyme crystallized as a intermediate, Ploux at al. [22] showed that this compound was a symmetrical homodimer, folded in three domains, the active site being potent slow binding competitive inhibitor of the B. sphaericus enzyme

Table 1 PLP-dependent α-oxoamine synthases related to 8-amino-7-oxononanoate synthase.

Enzyme EC number Reaction catalyzed

8-Amino-7-oxononanoate synthase EC 2.3.1.47 Pimeloyl-CoA +L-Ala→8-amino-7-oxononanoate+CO2 +CoASH

5-Aminolevulinate synthase EC 2.3.1.37 Succinyl-CoA +Gly→5-aminolevulinate+CO2 +CoASH

Serine palmitoyltransferase EC 2.3.1.50 Palmitoyl-CoA +L-Ser →3-dehydrosphinganine +CO2 +CoASH 2-Amino-3-oxobutyrate CoA ligasea EC 2.3.1.29 Acetyl-CoA+Gly→2-Amino-3-oxobutyrate +CoASH

a This synthase does not decarboxylate the intermediate and thus produces a 3-oxoacid. S. Mann, O. Ploux / Biochimica et Biophysica Acta 1814 (2011) 1459–1466 1461

Table 2 Steady-state kinetic parameters for the different AONSs so far studieda.

−1 −1 −1 −1 −1 AONS source KmAla (mM) KmPimeloyl-CoA (μM) kcat (s ) kcat/KmAla (M s ) kcat/KmPimeloyl-CoA (M s ) Ref. B. sphaericus 2.0 1.5 0.3 150 200 000 [8] E. coli 1.1 142 0.17 154 1197 [9] A. thaliana 1.4 1.6 0.1 78 62 500 [10] M. tuberculosis 0.5 1.5 0.004 15 2598 [11]

a The reported kinetic parameters are apparent parameters since they were determined from primary plots using a single finite concentration of the fixed substrate, rather than extrapolated from secondary plots using several concentrations of the fixed substrate.

(see below Section 2.4), a fact consistent with the proposed reaction the E. coli [9], the A. thaliana [10], and the M. tuberculosis [11] AONSs, mechanism. respectively) or using rapid kinetics (k=20000M−1 s−1 and −1 −1 More recently, using L-alanine methyl ester as the substrate for the E. k=399 M s for the E. coli [9] and M. tuberculosis [11] AONSs, coli AONS, Kerbarh et al. [23] showed that this analog formed a quinonoid respectively). The slow exchange of the C2-proton of L-alanine −1 intermediate in the absence of pimeloyl-CoA, a fact only compatible with observed on the B. sphaericus AONS [8] (kexch. =1.8 min , one the abstraction of the C2-proton. Furthermore, in the presence of tenth of the kcat) and structural considerations on the E. coli enzyme pimeloyl-CoA and L-alanine methyl ester they observed the accumulation [9], point to a flexible Ala-PLP aldimine and thus a C2–H bond not in of an intermediate which structure was deduced after sodium borohy- the correct position, that is parallel to the conjugated PLP π-orbital dride reduction and electrospray mass spectrometry analysis of the system, for rapid proton abstraction by the strictly conserved active enzyme species. The experimental mass observed corresponded to the site base lysine. mass of the apo-enzyme plus the mass of the reduced aldimine formed Formation of the Ala quinonoid. After binding of pimeloyl-CoA, between PLP and 8-amino-8-methyloxycarbonyl-7-oxononoate, the which induces a conformational change [8,9,22], the Ala quinonoid is intermediate that could not undergo decarboxylation. These data formed by abstraction of the C2-proton on the Si-face of the PLP confirmed unambiguously that AONS catalyzes the reaction using the aldimine by the active site lysine (the Si-face refers to the C4' mechanism depicted in Fig. 3. Since then, the reaction mechanism of KBL (carbonyl) prochiral trigonal carbon of the PLP). This Ala quinonoid [24] has been studied and it was concluded that all the α-oxoamine formation was observed by visible spectroscopy and rapid kinetics in synthases use the same reaction mechanism. the E. coli [9] and M. tuberculosis [11] AONSs, although the kinetics of The reaction mechanism can thus be described as follows (Fig. 3): its formation was complex. Formation of the Ala-PLP aldimine. The first step corresponds to the Formation of the intermediate. The acylation is thought to take place formation of the external aldimine with L-alanine, which has been with inversion of configuration, although this has never been directly studied by visible spectroscopy at equilibrium (Kd =4.1mM, proved. This stereochemical outcome is however consistent with data Kd =0.9 mM, Kd =1.8 mM, Kd =1.9 mM for the B. sphaericus [22], obtained on the related enzymes, ALAS and KBL. In the crystallized ALAS–succinyl-CoA complex, succinyl-CoA binds on the Re-face of the PLP aldimine [17], indirectly suggesting an inversion of configuration for the acylation step. In the case of KBL, that does not decarboxylate the intermediate, it has recently been shown that the acylation does involve an inversion of configuration [24]. Decarboxylation and reprotonation. The intermediate is then decarboxylated to yield the second quinonoid species. It is not yet known what is the position of the carbon-carbon bond before decarboxylation: either parallel to the PLP π-system or to the AON carbonyl π-orbitals, for facile rupture of the C-C bond (stereoelec- tronic principle). Reprotonation on the Si-face, likely catalyzed by the active site lysine, yields the AON external aldimine. This is consistent

with the observed exchange of the C8-proton of AON catalyzed by the B. sphaericus AONS [8] and with the three-dimensional structure of the E. coli AONS in complex with the AON product [9]. Product release. The AON product is then released by transaldimination by the active site lysine. The configuration of the AON product was proposed to be (S), based on the configuration of (+)-biotin, but it was only recently that Mann et al. [25] conclusively showed that it was the case. However, this compound racemizes quickly even at neutral pH [25]. Based on KIE measurements as well as kinetic parameters, it seems that the slowest step in this mechanism would be the reprotonation of the second quinonoid species, since a large solvent KIE has been observed [8]. Several arguments led to propose that the AONS changes its conformation during catalysis, and most likely upon pimeloyl-CoA Fig. 2. The three-dimensional structure of the E. coli holo-AONS is shown with that of three binding [8,9,22], which promotes a catalytic competent conformation, other representative α-oxoamine synthases, for comparison (only one monomer is or closed conformation. Although the description of this conforma- represented). The pictures were drawn using the RasMol software and the following tional change is not complete, structural and modeling studies coordinates retrieved from the Protein Data Bank (E. coli holo-AONS: 1DJE; Rhodobacter suggested that the C-terminal domain of the AONS moves during capsulatus holo-ALAS chain A: 2BWN; E. coli KBL in complex with 2-amino-3-oxobutyrate, chain A: 1FC4; Sphingomonas paucimobilis holo-SPT: 2JG2). α-Helices are colored in pink catalysis and that the hydrogen bond network around the PLP is also and β-strands in yellow. modified [9]. 1462 S. Mann, O. Ploux / Biochimica et Biophysica Acta 1814 (2011) 1459–1466

Fig. 3. Schematic representation of the active site structure of holo-AONS and the chemical reaction mechanism for this enzyme. The catalyzed reaction involves an acylation step with inversion of conﬁguration and a decarboxylation with retention of conﬁguration. This reaction mechanism also holds for the other α-oxoamine synthases.

2.4. Inhibitors of AONS enzyme tightly binds the intermediate. The best inhibitor is compound 1, a slow binding competitive inhibitor (one-step mechanism). This Only a few inhibitors of AONS have been described. Ploux et al. [22] compound forms an external aldimine with the B. sphaericus enzyme reported the inhibition of the B. sphaericus enzyme by D-alanine and that was detected by spectrophotometric titration [22]. Unfortunately, several analogs of the intermediate (Fig. 4). D-alanine inhibits AONS this complex could not be studied by X-ray crystallography. However, competitively with respect to L-alanine by forming an external the structure of this aldimine, formed with the E. coli AONS, has been aldimine that does not react further with pimeloyl-CoA, probably solved (O. Ploux, C. Cambillau, unpublished results). Surprisingly, the because the C2–H bond of D-alanine is not facing the active site base enantiomer bound is of the (S)-conﬁguration, the reverse of what was preventing proton abstraction. It is interesting to note that D-alanine expected based on the reaction mechanism. It is possible that this binds to the enzyme more tightly than L-alanine, probably because enantiomer binds more tightly to the active site than the other D-alanine is mimicking the tightly bound intermediate. enantiomer because of the favorable interaction of the phosphonate The synthetic analogs of the intermediate were all competitive group with His133. This interaction would not be possible with the inhibitors with respect to L-alanine [22]. They all had a better afﬁnity (R)-enantiomer because the phosphonate group would point toward than L-alanine (30 to 300 times higher) suggesting again that the the Si-face of the PLP system, as shown in Fig. 3 for the predicted

Fig. 4. A. Structure and inhibition constant for inhibitors of AONS [22,26]. B. The structure of the aldimine formed between compound 1 and the E. coli AONS is schematically represented here to show the configuration of the bound inhibitor (O. Ploux and C. Cambillau, unpublished results). S. Mann, O. Ploux / Biochimica et Biophysica Acta 1814 (2011) 1459–1466 1463 intermediate position. Thus, compound 1 probably inhibits the enzyme by mimicking the AON aldimine. Alexeev et al. [26] have reported the inactivation of the E. coli AONS by L-trifluoroalanine, a known suicide inactivator of PLP- dependent enzymes. The inactivation mechanism proposed is intriguing since it involves a decarboxylative elimination of fluorine rather than a β-elimination with C2-proton abstraction, as would be predicted by the reaction mechanism. The electrophilic conjugated system then probably reacts with the active site lysine to form a Fig. 5. The kinetic mechanism for DAPA AT and its inhibition by (R)-AON, the covalent bond, thus inactivating the enzyme. enantiomer of the substrate. (R)-AON is a competitive inhibitor with respect to both

substrates and binds to the two forms of the enzyme. The inhibition constants Ki1 and K were determined for the M. tuberculosis DAPA AT [25]. 3. 7,8-Diaminopelargonic acid aminotransferase i2

In the biosynthetic pathway to biotin, DAPA AT transforms the product of AONS to DAPA (Fig. 1). This aminotransferase activity was 3.2. Three-dimensional structure and reaction mechanism first detected in E. coli [27] and then in other bacteria [28]. The enzymes from Brevibacterium divaricatum [29] and from E. coli [30,31] were then The crystal structure of the E. coli DAPA AT in its holo-form and in purified and characterized. In 1999, the E. coli enzyme was crystallized complex with AON was reported in 1999 [33]. Since then, the and its three-dimensional structure determined [32,33].BecauseDAPA structure of DAPA AT from M. tuberculosis and from B. subtilis has been AT is inhibited by amiclenomycin, a natural antibiotic [34–38],and deposited to the Protein Data Bank. DAPA AT belongs to the PLP- because it is a potential therapeutic target in M. tuberculosis [39],the dependent aminotransferase I (fold I, subclass II) superfamily, and the enzyme from that pathogen was also carefully studied [25,40–42]. E. coli holo-enzyme crystallized as a homodimer, each monomer consisting of two domains. The PLP, which is located at the interface of 3.1. The reaction and the enzyme properties the two domains, is linked to Lys274 by an imine bond and to Asp245 by a hydrogen bond involving the protonated pyridine nitrogen. The DAPA AT catalyzes a simple transamination using PLP as a cofactor. determination of the structure, at 2.8 Å resolution, of the non- However, the amino donor is not an amino acid but S-adenosyl-L- productive complex with AON allowed the identification of the active methionine (AdoMet) [29,30], a unique situation among aminotrans- site residues in direct contact with AON. Overall, the AON molecule is ferases. The actual amino donor is the AdoMet with the (S)-configuration located at the Re-face of the PLP cofactor. The carboxylic group of AON at the sulfonium chiral center [43]. The product of the reaction, beside is linked to Arg391 by a salt bridge, the ketone group is linked to the DAPA is the α-ketoacid corresponding to AdoMet (S-adenosyl-2-oxo-4- Lys274 side chain nitrogen by a hydrogen bond, and the amine of AON methylthiobutyric acid). This compound is rather unstable and can is bonded to the phenolic hydroxyl of Tyr17 and to the peptide oxygen undergo β-elimination to give 5′-methylthioadenosine and 2-oxo-3,4- of Gly307. The aliphatic chain of AON is in direct van der Walls contact butenoic acid [30]. Using reductive conditions, Breen et al. [44] showed with the side chains of several aromatic residues: Tyr17, Trp53, indeed that the reduced product was detected (S-adenosyl-2-hydroxy-4- Tyr144 and Phe393. Recent experimental and computational work methylthiobutyric acid) confirming early the observation by Stoner and [25] suggested that the configuration of the bound AON, in the crystal Eisenberg [30]. Surprisingly, the enzyme from B. subtilis uses L-lysine or structure, is (R) rather that (S)asfirst proposed [33].

D-lysine rather than AdoMet as the amino donor, although the Km for Several mutants of the E. coli DAPA AT have been constructed, L-lysine is high (greater than 2 mM) [45].However,thissituationseemsto characterized and their structure determined, and it was shown that be unique among the DAPA AT so far studied. Tyr17, Tyr144, and Arg391 are involved in the binding of AON and in The kinetics of the reaction catalyzed by DAPA AT was carefully catalysis [43,47]. On the other hand, Arg253, which is outside of the studied on the E. coli enzyme [31] and then on the M. tuberculosis active site, seems to be involved in the binding of AdoMet [47]. enzyme [25,41] using a microbiological determination of DAPA Unfortunately, the structure of DAPA AT in complex with AdoMet or (Table 3). These steady state studies were extended, using the E. coli structural analogs has not been reported and thus it is not possible to DAPA AT, by measuring the kinetic parameters for the half reaction of describe the binding site of AdoMet. the PLP form of the enzyme with either AdoMet or DAPA as the The proposed reaction mechanism for the transamination, as substrate [43]. The kinetic mechanism is a Bi Bi ping-pong type and depicted in Fig. 6, involves the formation of the external aldimine with the enzyme is competitively inhibited by its substrate AON, with AdoMet and then the fast formation of the AdoMet quinonoid, which respect to AdoMet. Substrate inhibition is commonly observed in PLP- has been observed by visible spectroscopy and rapid kinetic dependent transaminases, but Mann et al. [25] using enantiopure AON techniques [43]. Reprotonation at the C4' position of the PLP by the recently reexamined this issue. It was shown that the DAPA AT from active site base, very likely the conserved lysine, followed by M. tuberculosis and that from E. coli were inhibited by (R)-AON the hydrolysis, gives the pyridoxamine-5′-phosphate (PMP)-form of enantiomer of the substrate but not by (S)-AON. In fact, (R)-AON DAPA AT. The second substrate (S)-AON can then enter the active binds to the two form of DAPA AT (Fig. 5). This situation could only be site and form the ketimine with the PMP. The lysine will again clariﬁed by using enantiopure AON that was only available using a catalyze the stereospeciﬁc aza-allylic isomerization to give the DAPA non-racemizing synthetic route [46]. external aldimine. The DAPA is then released by transaldimination.

Table 3 Steady-state kinetic parameters for the different DAPA ATs so far studied.

a −1 DAPA AT source KmAdoMet (μM) KmAON (μM) kcat (min ) KmPLP (μM) KmPMP (μM) Ref. E. coli 200 1.2 17 32 21 [31] M. tuberculosis 780 3.8 1 NDb ND [41]

a These values were determined using racemic AON. b Not determined. 1464 S. Mann, O. Ploux / Biochimica et Biophysica Acta 1814 (2011) 1459–1466

Fig. 6. Reaction mechanism for the transamination catalyzed by DAPA AT. The second half reaction, from AON ketimine to DAPA, is not represented since it is equivalent to the ﬁrst half reaction. R represents the AdoMet side chain.

3.3. Inhibition by amiclenomycin and substrate analogues step minimal kinetic scheme, as usually observed for irreversible inhibitors: Amiclenomycin is an amino acid produced by Streptomyces lavendulae that was isolated in 1974. Following its isolation a E+I⇄E&I→EÀI structure and a mode of action were proposed for this antibiotic −1 [34–38]. It was showed that this molecule inhibited the DAPA AT but with the following parameters: Ki =12μM and kinact. =0.35 min , the actual mechanism was not described. Interestingly, this antibiotic for the M. tuberculosis DAPA AT [41]. The partition ratio was close to was particularly active against M. tuberculosis but failed to cure unity indicating that this enzyme was inactivated by one molecule of infected mice. Another Streptomyces strain, S. venezuelae, produces amiclenomycin per active site, ranking amiclenomycin as a very peptides containing amiclenomycin that were effective against Gram- efficient suicide substrate. Inactivation by cis-amiclenomycin pro- negative bacteria, after uptake and enzymatic hydrolysis to produce voked a bleaching of the 425 nm absorption band indicating that the amiclenomycin [37,38]. reaction involved the PLP cofactor [50,51]. Determination of the three- Several years later, Mann et al. [48,49] proposed a total synthesis of dimensional structure of the inactivated E. coli DAPA AT [51] (Fig. 8)as cis- and trans-amiclenomycin (Fig. 7) and conclusively reattributed well as chemical identification of the final adduct by independent the configuration of natural amiclenomycin. Natural amiclenomycin synthesis and mass spectrometry analysis [52] showed that the has a cis-configuration for its disubstituted 6-membered cycle rather inactivation process led to an aromatized adduct that was tightly than a trans-configuration as first proposed. bound to the enzyme active site. The proposed inactivation mecha- The mode of action of this compound on the E. coli and M. nism, depicted in Fig. 9, involves a proton shift, as in the normal tuberculosis DAPA AT was then carefully studied and it was shown that catalytic cycle, yielding the PMP form of the enzyme. This is followed cis-amiclenomycin inactivated the enzyme in a time-dependent by an irreversible base-catalyzed tautomerization affording the fashion [50].Thetrans-isomer was much less active [50].The aromatic adduct [50]. This second step is very likely irreversible inactivation of the enzyme by cis-amiclenomycin followed a two- because it is an exergonic aromatization step. There is no obvious

Fig. 7. Structure of inhibitors of DAPA AT. S. Mann, O. Ploux / Biochimica et Biophysica Acta 1814 (2011) 1459–1466 1465

Fig. 8. Left. Schematic representation of the active site (Chain A) of E. coli DAPA AT inactivated by cis-amiclenomycin. The side chains of the amino acids that are involved in the binding of the adduct, are shown. Hydrogen bonds are represented as dashed lines. Right. The same active site drawn using the SwissPDB viewer software and the coordinates retrieved from the Protein Data Bank (accession 1MLY) [51]. The residues are colored as in the left hand side scheme. The residues bound to the phosphate group have been omitted for clarity. active site base close enough to remove the allylic proton, in this useful templates to design new molecules with potential herbicidal or irreversible step, and it was thus proposed that a water molecule antibacterial properties. could be the proton acceptor [51]. Several structural analogs of amiclenomycin were prepared and Acknowledgments tested as inactivators of DAPA AT. Mann et al. [40,41,50] found that fi while the cis-con guration of the cycle was required to observe We thank all our collaborators and students that were involved in inactivation, the amino acid moiety was not essential. Compound 4, the biotin biosynthesis project. the enantiomer of cis-amiclenomycin amide, and the cis-analogs (compounds 5 and 6) lacking the amino acid functionality were all inactivators. Interestingly, compound 6 was active in vivo by References μ inhibiting the growth of M. smegmatis at 10 g/mL [41]. Thus simple [1] M.A. Eisenberg, Biotin: biogenesis, transport, and their regulation, Adv. Enzymol. analogs of amiclenomycin might be useful as template for the design Relat. Areas Mol. Biol. 38 (1973) 317–372. of new inactivators of DAPA AT. [2] S. Lin, R.E. Hanson, J.E. Cronan, Biotin synthesis begins by hijacking the fatty acid synthetic pathway, Nat. Chem. Biol. 6 (2010) 682–688. More recently, the analog of AON lacking the methyl group, 8- [3] O. Ploux, P. Soularue, A. Marquet, R. Gloeckler, Y. Lemoine, Investigation of the first amino-7-oxooctanoic acid, was prepared and shown to be active as an step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization herbicide [53]. Mann et al. [25] showed that this compound had the of the pimeloyl-CoA synthase, and uptake of pimelate, Biochem. J. 287 (1992) – same behavior than (R)-AON and inhibited the M. tuberculosis DAPA 685 690. [4] M.A. Eisenberg, C. Star, Synthesis of 7-oxo-8-aminopelargonic acid, a biotin AT, by binding to the two forms of the enzyme with the following vitamer, in cell-free extracts of Escherichia coli biotin auxotrophs, J. Bacteriol. 96 inhibition constants: Ki1 =4.2μM, and Ki2 =0.9 μM. This simple (1968) 1291–1297. scaffold might be useful for the design of new inhibitors of this [5] Y. Izumi, K. Sato, Y. Tani, K. Ogata, Distribution of 7-keto-8-aminopelargonic acid synthetase in bacteria and the control mechanism of the enzyme activity, Agric. enzyme. Biol. Chem. 37 (1973) 1335–1340. [6] Y. Izumi, H. Morita, Y. Tani, K. Ogata, Partial purification and some properties of 7- keto-8-aminopelargonic acid synthetase, an enzyme involved in biotin biosyn- – 4. Conclusions and perspectives thesis, Agric. Biol. Chem. 37 (1973) 1327 1333. [7] O. Ploux, A. Marquet, The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme The biosynthesis of biotin has been studied for many years but the overproduced in Escherichia coli, Biochem. J. 283 (1992) 327–331. detailed description of the enzymatic steps involved in this pathway [8] O. Ploux, A. Marquet, Mechanistic studies on the 8-amino-7-oxopelargonate synthase, a pyridoxal-5′-phosphate-dependent enzyme involved in biotin has only been possible recently thanks to the constant efforts of biosynthesis, Eur. J. Biochem. 236 (1996) 301–308. several groups. Among the four steps leading to biotin from pimeloyl- [9] S.P. Webster, D. Alexeev, D.J. Campopiano, R.M. Watt, M. Alexeeva, L. Sawyer, R.L. CoA, two are catalyzed by PLP-dependent enzymes, the AONS and the Baxter, Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, – DAPA AT. The structure as well as the reaction mechanism of these and crystallographic studies, Biochemistry 39 (2000) 516 528. [10] V. Pinon, S. Ravanel, R. Douce, C. Alban, Biotin synthesis in plants. The first two enzymes is now well described. Furthermore, several interesting committed step of the pathway is catalyzed by a cytosolic 7-keto-8-aminope- inhibitors have been designed and characterized and might serve as largonic acid synthase, Plant. Physiol. 139 (2005) 1666–1676.

Fig. 9. Schematic representation of the inactivation mechanism of DAPA AT by cis-amiclenomycin and its analogs. R=(CH2)2–CHNH2–COOH for amiclenomycin. 1466 S. Mann, O. Ploux / Biochimica et Biophysica Acta 1814 (2011) 1459–1466

[11] V.M. Bhor, S. Dev, G.R. Vasanthakumar, P. Kumar, S. Sinha, A. Surolia, Broad substrate [32] H. Kack, K.J. Gibson, A.A. Gatenby, G. Schneider, Y. Lindqvist, Purification and stereospecificity of the Mycobacterium tuberculosis 7-keto-8-aminopelargonic acid preliminary X-ray crystallographic studies of recombinant 7,8-diaminopelargonic synthase: spectroscopic and kinetic studies, J. Biol. Chem. 281 (2006) 25076–25088. acid synthase from Escherichia coli, Acta Crystallogr. D 54 (1998) 1397–1398. [12] T. Kubota, J. Shimono, C. Kanameda, Y. Izumi, The first thermophilic alpha-oxoamine [33] H. Kack, J. Sandmark, K. Gibson, G. Schneider, Y. Lindqvist, Crystal structure of synthase family enzyme that has activities of 2-amino-3-ketobutyrate CoA ligase and diaminopelargonic acid synthase: evolutionary relationships between pyridoxal- 7-keto-8-aminopelargonic acid synthase: cloning and overexpression of the gene 5′-phosphate-dependent enzymes, J. Mol. Biol. 291 (1999) 857–876. from an extreme thermophile, Thermus thermophilus, and characterization of its gene [34] Y. Okami, T. Kitahara, M. Hamada, H. Naganawa, S. Kondo, Studies on a new amino product, Biosci. Biotechnol. Biochem. 71 (2007) 3033–3040. acid antibiotic, amiclenomycin, J. Antibiot. 27 (1974) 656–664. [13] S. Spinelli, O. Ploux, A. Marquet, C. Anguille, C. Jelsch, C. Cambillau, C. Martinez, [35] K. Hotta, T. Kitahara, Y. Okami, Studies of the mode of action of amiclenomycin, J. Crystallization and preliminary X-ray study of the 8-amino-7-oxopelargonate Antibiot. 28 (1975) 222–228. synthase from Bacillus sphaericus, Acta Crystallogr. D 52 (1996) 866–868. [36] T. Kitahara, K. Hotta, M. Yoshida, Y. Okami, Biological studies of amiclenomycin, J. [14] D. Alexeev, M. Alexeeva, R.L. Baxter, D.J. Campopiano, S.P. Webster, L. Sawyer, The Antibiot. 28 (1975) 215–221. crystal structure of 8-amino-7-oxononanoate synthase: a bacterial PLP-depen- [37] A. Kern, U. Kabatek, G. Jung, R.G. Werner, M. Poetsch, H. Zähner, Amiclenomycin dent, acyl-CoA-condensing enzyme, J. Mol. Biol. 284 (1998) 401–419. peptides—isolation and structure elucidation of new biotin antimetabolites, [15] M.D. Toney, E. Hohenester, S.W. Cowan, J.N. Jansonius, Dialkylglycine decarboxylase Liebigs Ann. Chem. (1985) 877–1098. structure: bifunctional active site and alkali metal sites, Science 261 (1993) 756–759. [38] M. Poetsch, H. Zahner, R.G. Werner, A. Kern, G. Jung, Metabolic products from [16] A. Schmidt, J. Sivaraman, Y. Li, R. Larocque, J.A. Barbosa, C. Smith, A. Matte, J.D. microorganisms. 230. Amiclenomycin-peptides, new antimetabolites of biotin. Schrag, M. Cygler, Three-dimensional structure of 2-amino-3-ketobutyrate CoA Taxonomy, fermentation and biological properties, J. Antibiot. 38 (1985) 312–320. ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred [39] J. Keer, M.J. Smeulders, K.M. Gray, H.D. Williams, Mutants of Mycobacterium smegmatis reaction mechanism, Biochemistry 40 (2001) 5151–5160. impaired in stationary-phase survival, Microbiology 146 (2000) 2209–2217. [17] I. Astner, J.O. Schulze, J. van den Heuvel, D. Jahn, W.D. Schubert, D.W. Heinz, [40] S. Mann, A. Marquet, O. Ploux, Inhibition of 7,8-diaminopelargonic acid Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme aminotransferase by amiclenomycin and analogues, Biochem. Soc. Trans. 33 biosynthesis, and its link to XLSA in humans, EMBO J. 24 (2005) 3166–3177. (2005) 802–805. [18] B.A. Yard, L.G. Carter, K.A. Johnson, I.M. Overton, M. Dorward, H. Liu, S.A. [41] S. Mann, O. Ploux, 7, 8-Diaminoperlargonic acid aminotransferase from McMahon, M. Oke, D. Puech, G.J. Barton, J.H. Naismith, D.J. Campopiano, The Mycobacterium tuberculosis, a potential therapeutic target. Characterization and structure of serine palmitoyltransferase; gateway to sphingolipid biosynthesis, J. inhibition studies, FEBS J. 273 (2006) 4778–4789. Mol. Biol. 370 (2007) 870–886. [42] V.M. Bhor, S. Dev, G.R. Vasanthakumar, A. Surolia, Spectral and kinetic [19] M.M. Abboud, P.M. Jordan, M. Akhtar, Biosynthesis of 5-aminolevulinic acid: characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium involvement of a retention–inversion mechanism, J. Chem. Soc. Chem. Comm. tuberculosis, IUBMB Life 58 (2006) 225–233. (1974) 643–644. [43] A.C. Eliot, J. Sandmark, G. Schneider, J.F. Kirsch, The dual-specific active site of 7,8- [20] J. Zhang, G.C. Ferreira, Transient state kinetic investigation of 5-aminolevulinate diaminopelargonic acid synthase and the effect of the R391A mutation, synthase reaction mechanism, J. Biol. Chem. 277 (2002) 44660–44669. Biochemistry 41 (2002) 12582–12589. [21] K. Krisnangkura, C.C. Sweeley, Studies on the mechanism of 3-ketosphinganine [44] R.S. Breen, D.J. Campopiano, S. Webster, M. Brunton, R. Watt, R.L. Baxter, The synthetase, J. Biol. Chem. 251 (1976) 1597–1602. mechanism of 7,8-diaminopelargonate synthase; the role of S-adenosylmethio- [22] O. Ploux, O. Breyne, S. Carillon, A. Marquet, Slow-binding and competitive nine as the amino donor, Org. Biomol. Chem. 1 (2003) 3498–3499. inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5′-phosphate- [45] S.W. Van Arsdell, J.B. Perkins, R.R. Yocum, L. Luan, C.L. Howitt, N.P. Chatterjee, J.G. dependent enzyme involved in biotin biosynthesis, by substrate and intermediate Pero, Removing a bottleneck in the Bacillus subtilis biotin pathway: bioA utilizes analogs. Kinetic and binding studies, Eur. J. Biochem. 259 (1999) 63–70. lysine rather than S-adenosylmethionine as the amino donor in the KAPA-to- [23] O. Kerbarh, D.J. Campopiano, R.L. Baxter, Mechanism of alpha-oxoamine DAPA reaction, Biotechnol. Bioeng. 91 (2005) 75–83. synthases: identification of the intermediate Claisen product in the 8-amino-7- [46] D. Lucet, T. Le Gall, C. Mioskowski, O. Ploux, A. Marquet, First synthesis of both oxononanoate synthase reaction, Chem. Commun. (2006) 60–62. enantiomers of the biotin vitamer 8-amino-7-oxopelargonic acid, Tetrahedron [24] Q. Bashir, N. Rashid, M. Akhtar, Mechanism and substrate stereochemistry of 2- Asymmetr. 7 (1996) 985–988. amino-3-oxobutyrate CoA ligase: implications for 5-aminolevulinate synthase [47] J. Sandmark, A.C. Eliot, K. Famm, G. Schneider, J.F. Kirsch, Conserved and and related enzymes, Chem. Commun. (2006) 5065–5067. nonconserved residues in the substrate binding site of 7,8-diaminopelargonic [25] S. Mann, L. Colliandre, G. Labesse, O. Ploux, Inhibition of 7,8-diaminopelargonic acid synthase from Escherichia coli are essential for catalysis, Biochemistry 43 acid aminotransferase from Mycobacterium tuberculosis by chiral and achiral (2004) 1213–1222. anologs of its substrate: biological implications, Biochimie 91 (2009) 826–834. [48] S. Mann, S. Carillon, O. Breyne, A. Marquet, Total synthesis of amiclenomycin, an [26] D. Alexeev, R.L. Baxter, D.J. Campopiano, O. Kerbarh, L. Sawyer, N. Tomczyk, R. inhibitor of biotin biosynthesis, Chem. Eur. J. 8 (2002) 439–450. Watt, S.P. Webster, Suicide inhibition of alpha-oxamine synthases: structures of [49] S. Mann, S. Carillon, O. Breyne, C. Duhayon, L. Hamon, A. Marquet, Synthesis and the covalent adducts of 8-amino-7-oxononanoate synthase with trifluoroalanine, stereochemical assignments of cis- and trans-1-amino-4-ethylcyclohexa-2,5- Org. Biomol. Chem. 4 (2006) 1209–1212. diene as models for amiclenomycin, Eur. J. Org. Chem. (2002) 736–744. [27] C.H. Pai, Biosynthesis of biotin: synthesis of 7,8-diaminopelargonic acid in cell- [50] S. Mann, D. Florentin, D. Lesage, T. Drujon, O. Ploux, A. Marquet, Inhibition of free extracts of Escherichia coli, J. Bacteriol. 105 (1971) 793–800. diamino pelargonic acid aminotransferase, an enzyme of the biotin biosynthetic [28] Y. Izumi, K. Sato, Y. Tani, K. Ogata, 7, 8-diaminopelargonic acid aminotransferase, pathway, by amiclenomycin: a mechanistic study, Helv. Chim. Acta 86 (2003) an enzyme involved in biotin biosynthesis by microorganisms, Agric. Biol. Chem. 3836–3850. 39 (1975) 175–181. [51] J. Sandmark, S. Mann, A. Marquet, G. Schneider, Structural basis for the inhibition [29] Y. Izumi, K. Sato, Y. Tani, K. Ogata, Purification of 7-Keto-8-aminopelargonic acid-7, 8- of the biosynthesis of biotin by the antibiotic amiclenomycin, J. Biol. Chem. 277 diaminopelargonic acid aminotransferase, an enzyme involved in biotin biosynthe- (2002) 43352–43358. sis, from Brevibacterium divaricatum, Agric. Biol. Chem. 37 (1973) 2683–2684. [52] S. Mann, D. Lesage, J.C. Tabet, A. Marquet, Identification of the products of reaction [30] G.L. Stoner, M.A. Eisenberg, Purification and properties of 7,8-diaminopelargonic between pyridoxal phosphate and amiclenomycin and other related 1-amino- acid aminotransferase, J. Biol. Chem. 250 (1975) 4029–4036. cyclohexa-2,5-dienes, Tetrahedron 59 (2003) 5209–5214. [31] G.L. Stoner, M.A. Eisenberg, Biosynthesis of 7, 8-diaminopelargonic acid from 7- [53] A. Nudelman, D. Marcovici-Mizrahi, A. Nudelman, D. Flint, V. Wittenbach, keto-8-aminopelargonic acid and S-adenosyl-L-methionine. The kinetics of the Inhibitors of biotin biosynthesis as potential herbicides, Tetrahedron 60 (2004) reaction, J. Biol. Chem. 250 (1975) 4037–4043. 1731–1748.