Structural Basis for the Inhibition of the Biosynthesis of Biotin by the Antibiotic Amiclenomycin

Abstract

The antibiotic amiclenomycin blocks the biosynthesis of biotin by inhibiting the pyridoxal-phosphate-dependent enzyme diaminopelargonic acid synthase. Inactivation of the enzyme is stereoselective, i.e. the cis isomer of amiclenomycin is a potent inhibitor, whereas the trans isomer is much less reactive. The crystal structure of the complex of the holoenzyme and amiclenomycin at 1.8 Å resolution reveals that the internal aldimine linkage between the cofactor and the side chain of the catalytic residue Lys-274 is broken. Instead, a covalent bond is formed between the 4-amino nitrogen of amiclenomycin and the C4 carbon atom of pyridoxal-phosphate. The electron density for the bound inhibitor suggests that aromatization of the cyclohexadiene ring has occurred upon formation of the covalent adduct. This process could be initiated by proton abstraction at the C4 carbon atom of the cyclohexadiene ring, possibly by the proximal side chain of Lys-274, leading to the tautomer Schiff base followed by the removal of the second allylic hydrogen. The carboxyl tail of the amiclenomycin moiety forms a salt link to the conserved residue Arg-391 in the substrate-binding site. Modeling suggests steric hindrance at the active site as the determinant of the weak inhibiting potency of the trans isomer.The biosynthesis of the vitamin biotin, a cofactor in biological carboxylation reactions, occurs in micro-organisms and plants and involves at least four different steps (Scheme 1) (1-12). The second step in this pathway, the conversion of 7-keto-8-aminopelargonic acid (KAPA) 1 into 7,8-diaminopelargonic acid (DAPA), is catalyzed by DAPA synthase, an aminotransferase that requires PLP as a cofactor (6, 7). DAPA synthase from Escherichia coli is a homodimer with a molecular mass of 94 kDa (6, 13) and contains 429 residues per monomer (14). The enzyme is unique among aminotransferases in that it uses S-adenosyl-L-methionine (SAM) as an amino group donor. The reaction catalyzed by DAPA synthase is typical of that of an aminotransferase and follows the general mechanism seen in other PLP-dependent enzymes (15).The crystal structure of DAPA synthase (16) shows that DAPA synthase belongs to the fold type I family of PLPdependent enzymes (17, 18). The monomer of DAPA synthase consists of two domains, a small domain comprising the N-and C-terminal part of the polypeptide chain (residues 1-49 and 330 -429) and a large domain formed by the intervening residues containing the cofactor-binding site. In the crystal, the two subunits of the homodimer of DAPA synthase are related by a 2-fold non-crystallographic axis (16). The PLP-binding site is located between the two domains of the monomer at the interface of the two subunits. In the enzyme-PLP complex, the cofactor is covalently linked to the ⑀-amino group of Lys-274, a residue functionally invariant in the whole family of fold-type I PLP-dependent enzymes.Because biotin synthesis is unique to plants and microorganisms, enzymes of this pathway are potential targets for the development of antimicr...

Inhibition of 7,8-diaminopelargonic acid aminotransferase by amiclenomycin and analogues

“…Inactivation of Escherichia coli DAPA AT by amiclenomycin and analogues was carefully studied using different approaches and was shown to be irreversible with the formation of an aromatic PLP-amiclenomycin adduct 2 that is tightly bound to the active site (Scheme 3) [8][9][10]. The natural cis-amiclenomycin 1a was much more efficient than the trans compound 1b, confirming our stereochemical assignment.…”

Pyridoxal 5-Phosphate Enzymes as Targets for Therapeutic Agents

“…Inhibition studies of E. coli DAPAS by amiclenomycin indicated that the inhibitor binds to the 7-keto-8-aminopelargonic acid/7,8-diaminopelargonic acid binding site of the enzyme [421]. The crystal structures of the complexes formed between the E. coli holoenzyme and cis and trans amiclenomycin have been determined [422]. The structure analysis revealed that the inhibitor forms a covalent adduct with the cofactor PLP, which mimics the external aldimine.…”

“…glycine acid) [391] 1K7F, 1.90 (N-[1H-indol-3-yl-acetyl] valine acid) [391] 7,8-diaminopelargonic acid synthase (I) 1QJ5, 1.80 [417] 1QJ3, 2.70 (7-keto-8-aminopelargonic acid) [417] 1MLZ, 2.15 (trans-amiclenomycin) [422] 1MLY, 1.81 (cis-amiclenomycin) [422] 8-amino-7-oxopelargonate synthase (I) 1BS0, 1.65 [429] 1DJ9, 2.00 ((S)-8-amino-7-oxonanonoate) [432] 2G6W, 2.14 (trifluoroalanine adduct) [433] 1DJE, 1.71 [429] early in the evolution (before the three biological kingdoms diverged) from different protein ancestors which generated five independent families, each corresponding to a different fold type [20][21][22]. The families have been named from the more representative enzyme.…”

Biotin biosynthesis in Mycobacterium tuberculosis: physiology, biochemistry and molecular intervention

“…The BioA inhibitor, amiclenomycin, is a narrow-spectrum antibiotic with activity against Mycobacteria sp., but not other bacteria or fungi that can scavenge exogenous biotin (Kitahara et al, 1975). Its anti-TB activity can be reversed by high concentrations of external biotin, above 0.01 μg/mL (Sandmark et al, 2002;Mann et al, 2005), which is at least 10-fold greater than the concentration found in normal human plasma (Mock and Malik, 1992). This implies that the water-soluble biotin might enter through the bacilli membrane using mechanisms that are not yet identified, but only in supra-physiological concentrations of the nutrient.…”