Convergent biosynthetic pathways to β-lactam antibiotics

Abstract

Five naturally-occurring families of β-lactams have inspired a class of drugs that constitute >60% of the antimicrobials used in human medicine. Their biosynthetic pathways reveal highly individualized synthetic strategies that yet converge on a common azetidinone ring assembled in structural contexts that confer selective binding and inhibition of d,d-transpeptidases that play essential roles in bacterial cell wall (peptidoglycan) biosynthesis. These enzymes belong to a single 'clan' of evolutionarily distinct serine hydrolases whose active site geometry and mechanism of action is specifically matched by these antibiotics for inactivation that is kinetically competitive with their native function. Unusual enzyme-mediated reactions and catalytic multitasking in these pathways are discussed with particular attention to the diverse ways the β-lactam itself is generated, and more broadly how the intrinsic reactivity of this core structural element is modulated in natural systems through the introduction of ring strain and electronic effects.

Figure 1

Representative structures of the five known subclasses of β-lactam antibiotics.

Figure 2

Left, asparagine synthetase, class B from E. coli. Its crystal structure [8] is shown and the reaction catalyzed is depicted beneath. Glutaminase domain (upper) is in purple with Gln bound (green CPK). The N-terminal catalytic Cys is replaced with Ala (red ball-and-stick). The synthetase domain (lower) in yellow has AMP bound (light blue CPK) and the conserved active site Glu is shown (red ball-and-stick). Right, β-lactam synthetase from S. clavuligerus. Its x-ray structure is shown and reaction catalyzed in clavulanic acid (1) biosynthesis is placed directly beneath. The corresponding carbapenam synthetase (CPS) reaction active in carbapenem-3-carboxylate (14) biosynthesis is drawn just below that. The non-functional glutaminase domain (upper) in purple with the Phe and nine additional N-terminal residues shown (purple CPK). The isolated synthetase domain (lower) in yellow has carboxyethyl-L-arginine (CEA) (green CPK) and AMP–CPP (light blue CPK) bound in the active site with the Tyr•Glu catalytic dyad highlighted (red ball-and-stick).

Figure 3

(a) Overview of the clavam (11) and clavulanic acid (1) pathways. (b) Overview of the “simple” carbapenem pathway to carbapenem-3-carboxylate (14) in Gram-negative enterobacteria and the “complex” pathway to thienamycin (2) in Gram-positive actinobacteria.

Figure 4

Module and domain organization of L-δ-(α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and the stepwise oxidative cyclization of ACV to isopenicillin N (3) by isopenicillin N synthase. After epimerization of the N-terminus of 3 from L- to D-, successive α-KG dependent non-heme iron enzymes act to carry out oxidative ring expansion to the cephem nucleus, allylic oxidation and, finally, oxidation and O-methylation at C-7 to give cephamycins.

Figure 5

(a) Summary of whole-cell incorporation experiments to establish the amino acid building blocks of nocardicin A (5). (b) Model reaction of β-lactam formation under Mitsunobu conditions gave a 3:2 mixture of diasteromeric products shown (Ft = N-phthaloyl, DEAD = diethyl diazodicarboxylate, Bn = benzyl). (c) Module and domain organization of NocA and NocB with NocI bound in trans to A1, A2 and A4, which need this MbtH superfamily protein to observe PPi exchange in vitro. The predicted product of canonical linear NRPS synthesis is L-pHPG–L-Arg–D-pHPG–L-Ser–L-pHPG (20).

Figure 6

(a) Tripeptide and (b) pentapeptide N-acetylcysteamine (SNAC) and S-pantethienyl (SPant) mimicks of the potential peptide products of NocA/B tested for reaction in the thioesterase (TE) domain. (c) In the course of preparing the tripeptides 29, 30 and 31 directly bound to the module 5 peptidyl carrier-domain–thioesterase didomain (PCP5–TE), epimerization at the C-terminus was observed and unavoidable.

Figure 7

(a) Diastereomeric D,L,L- and D,L,D-tripeptides and L,L,D,L,L- and L,L,D,L,D-pentapeptides that contain an embedded β-lactam rig in place of a seryl residue activated as substrate-like thioesters for possible reaction in the TE. (b) Proposed mechanism of β-lactam formation in the condensation (C) domain of module 5 followed by C-terminal epimerization (L- to D-) and hydrolytic product release.