Biochimica et Biophysica Acta 1814 (2011) 1459–1466 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap 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 1570-9639/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2010.12.004 1460 S. Mann, O. Ploux / Biochimica et Biophysica Acta 1814 (2011) 1459–1466 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.