beta-Lactam synthetase: a new biosynthetic enzyme

Abstract

The principal cause of bacterial resistance to penicillin and other beta-lactam antibiotics is the acquisition of plasmid-encoded beta-lactamases, enzymes that catalyze hydrolysis of the beta-lactam bond and render these antibiotics inactive. Clavulanic acid is a potent inhibitor of beta-lactamases and has proven clinically effective in combating resistant infections. Although clavulanic acid and penicillin share marked structural similarities, the biosyntheses of their bicyclic nuclei are wholly dissimilar. In contrast to the efficient iron-mediated oxidative cyclization of a tripeptide to isopenicillin N, the critical beta-lactam ring of clavulanic acid is demonstrated to form by intramolecular closure catalyzed by a new type of ATP/Mg2+-dependent enzyme, a beta-lactam synthetase (beta-LS). Insertional inactivation of its encoding gene in wild-type Streptomyces clavuligerus resulted in complete loss of clavulanic acid production and the accumulation of N2-(carboxyethyl)-L-arginine (CEA). Chemical complementation of this blocked mutant with authentic deoxyguanidinoproclavaminic acid (DGPC), the expected product of the beta-LS, restored clavulanic acid synthesis. Finally, overexpression of this gene gave the beta-LS, which was shown to mediate the conversion of CEA to DGPC in the presence of ATP/Mg2+. Primary amino acid sequence comparisons suggest that this mode of beta-lactam formation could be more widely spread in nature and mechanistically related to asparagine synthesis.

Figure 1

Reaction of isopenicillin N synthase, an overview of clavulanic acid biosynthesis and representative members of the β-lactam antibiotic classes. In penicillin biosynthesis, δ-(l-aminoadipoyl)-l-cysteinyl-d-valine (ACV) (1) is cyclized to isopenicillin N (2) by isopenicillin N synthase in the presence of ferrous ion and molecular oxygen. Clavulanic acid biosynthesis differs in that N²-(2-carboxyethyl)arginine (CEA) (3) cyclizes first to the monocyclic β-lactam deoxyguanidinoproclavaminate (DGPC) (4) mediated by β-lactam synthetase (β-LS) and ATP/Mg²⁺. Several subsequent transformations of this molecule are required to form clavulanic acid (5). The penam/cephem group is represented here by isopenicillin N (2), the clavams by clavulanic acid (5), the monocyclic β-lactams by nocardicin A (6), and the carbapenems by carbapenem-3-carboxylic acid (7).

Figure 2

(a) Restriction map showing the predicted result of a double crossover between homologous regions of orf3 in pLRF25 and the S. clavuligerus chromosome. Solid box represents the tsr gene, open box represents the neomycin resistance gene, and the crosshatched box represents orf3. Abbreviations: B, BclI; C, ClaI; E, EcoRI; Ev, EcoRV; H, HindIII; K, KpnI; N, NcoI; P, PvuII; Sa, SacI; Sp, SphI. (b) Southern hybridization of S. clavuligerus chromosomal DNA from RFL35 and wild type with orf3-specific probe (probe 1) and tsr-specific probe (probe 2, after striping gel of probe 1). Lanes: 1, RFL35/BclI; 2, WT/BclI; 3, RFL35/SphI; 4, WT/SphI; 5, RFL35/KpnI–SacI; 6, WT/KpnI–SacI; Mr, λ/HindIII.

Figure 3

Colorimetric assays. Accumulated intermediates in RFL35 vs. wild-type (WT) strains are evident by Sakaguchi color reaction in A. (B) Chemical complementation of RFL35 with deoxyguanidinoproclavaminate, visible by imidazole assay in as little as 24 hr.

Figure 4

Alignment of homologous regions of AS and β-LS. Heavy shading is used to indicate conserved residues and light shading is used to indicate similarity. The first and second regions are closely linked to glutamine-dependent activity in AS, and the third region is associated with ATP binding. Human AS (human AS P08243), Rice AS (rice AS U55873), Myco tuber. AS (Mycobacterium tuberculosis), Eco K12 AS-B (Q10374 E. coli P22106), β-LS (β-LS from S. clavuligerus), CarA (CarA from Erwinia cartovora).

Figure 5

Proposed mechanism of β-LS based on analogy to AS-B and β-lactamase.