b-Lactam synthetase: A new biosynthetic enzyme (b-lactam antibioticsybiosynthesisyclavulanic acidyamidotransferases)

The principal cause of bacterial resistance to penicillin and other b-lactam antibiotics is the acquisition of plasmid-encoded b-lactamases, enzymes that catalyze hydrolysis of the b-lactam bond and render these antibiotics inactive. Clavulanic acid is a potent inhibitor of b-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 b-lactam ring of clavulanic acid is demonstrated to form by intramolecular closure catalyzed by a new type of ATPyMg21-dependent enzyme, a b-lactam synthetase (b-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 b-LS, restored clavulanic acid synthesis. Finally, overexpression of this gene gave the b-LS, which was shown to mediate the conversion of CEA to DGPC in the presence of ATPyMg21. Primary amino acid sequence comparisons suggest that this mode of b-lactam formation could be more widely spread in nature and mechanistically related to asparagine synthesis. Penicillin and related b-lactam antibiotics have been a mainstay in the treatment of infections for 50 years. Their effectiveness in medicine, however, like other classes of known antibiotics, has come under increasing challenge from the rise of multiply drug-resistant pathogenic bacteria (1, 2). Prominant among the resistance mechanisms to penicillin and other b-lactams are the b-lactamases, which hydrolyze these antibiotics and disarm their ability to inhibit bacterial cell wall biosynthesis. Clavulanic acid is a naturally occurring inhibitor of b-lactamase enzymes, whose value in combating antibiotic resistance has been demonstrated clinically (3). A new mechanism of b-lactam biosynthesis has been identified in the clavulanate pathway, which may play an analogous role in the formation of the carbapenems, another clinically important family of these antibiotics. Application of these enzymes in the preparation of these and other antibiotics may be envisioned. As largely the products of fermentation, mechanistic and practical interests have given impetus to the study and genetic manipulation of antibiotic biosynthesis (4–7). Great effort has been focused on understanding the formation of these important natural products and to identifying the genes that encode their biosynthetic pathways. The b-lactam antibiotic family can be divided into at least four known groups, of which the biosynthetic study of penicillin N (Fig. 1, 2) is by far the most advanced. A tripeptide precursor 1 is produced by a nonribosomal peptide synthetase (8, 9), which is cyclized with impressive efficiency by isopenicillin N synthase in the presence of ferrous ion and molecular oxygen to isopenicillin N (Fig. 1, 2; subsequent boldface numbers will refer to corresponding sections of figure). Research has culminated recently in the x-ray crystal structure of isopenicillin N synthase with iron and substrate bound (10). The structural features of this complex, in addition to a vast base of earlier experimental data, have led to the proposal of a detailed catalytic mechanism involving successive oxidative cyclization reactions mediated by iron and oxygen resulting in the formation of two rings and two molecules of water (10, 11). The highly efficacious b-lactamase inhibitor clavulanic acid (5) bears strong structural similarity to isopenicillin N (2), but its primary modes of biochemical activity and synthesis differ sharply (3). Unlike the economical two-step biosynthesis of isopenicillin N (2), the fused bicyclic motif in clavulanic acid is assembled in at least eight steps from the building blocks of primary metabolism. The earliest proposed intermediate in clavulanic acid biosynthesis is CEA (3) (12, 13). An isotopically labeled sample of this material was shown to give an intact, albeit low, incorporation into 5 when administered to a fermentation of Streptomyces clavuligerus. Similarly, the monocyclic b-lactam 4 (DGPC) was prepared in labeled form and analogously incorporated into clavulanic acid, suggesting a precursor relationship of the former to the latter. In this paper we describe the first evidence that the transformation of CEA (3) to DGPC (4) is catalyzed by a single enzyme in the presence of ATPyMg21. This intramolecular amide bond formation is previously unknown and has led to the designation of a b-LS. The b-LS is encoded by orf3, a component of the clavulanic acid gene cluster (14) having deduced amino acid similarity to a subset of the amidotransferases (15). It is located approximately 1 kb upstream of orf5, which encodes clavaminate synthase (16), a later enzyme in the pathway responsible for formation of the bicyclic nucleus of clavulanic acid (17). Targeted disruption of orf3 in the wild-type S. clavuligerus led to complete blocking of clavulanic acid production and the accumulation of CEA. Chemical complementation of orf3 mutants with authentic DGPC was shown to restore clavulanic acid production. Finally, separate overexpression of orf3 afforded b-LS, which was demonstrated to catalyze the in vitro conversion of CEA (3) to DGPC (4) in the presence of ATPyMg21. EXPERIMENTAL PROCEDURES Gene Disruption. All restriction enzymes were purchased from New England Biolabs. Plasmid pLRF16 contained a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y959082-5$2.00y0 PNAS is available online at www.pnas.org. Abbreviations: b-LS, b-lactam synthetase; CEA, N2-(carboxyethyl)L-arginine; DGPC, deoxyguanidinoproclavaminic acid; AS-B, asparagine synthetase, Class-B. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF071051). *To whom reprint requests should be addressed. e-mail: Townsend@ jhunix.hcf.jhu.edu.