Three Unusual Reactions Mediate Carbapenem and Carbapenam Biosynthesis

Li, Rongfeng; Stapon, Anthony; Blanchfield, Joanne T.; Townsend, Craig A.

doi:10.1021/ja001723l

Cited by 63 publications

(85 citation statements)

References 21 publications

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“…Similar clusters have since been found in both the opportunistic human pathogen Serratia marcescens (5) and the entomopathogenic bacterium Photorhabdus luminescens (7). Nine genes comprise the cluster; five of them are structural genes (carABCDE), although only three genes (carABC) are absolutely required for carbapenem-3-carboxylic acid (Car) biosynthesis (18,23): CarB catalyzes the first step in the pathway, generating the pyrrolidine ring after precursor condensation; CarA catalyzes the formation of the ␤-lactam ring; and, finally, CarC converts the carbapenam to carbapenem (for details, see reference 4). CarD and CarE were proposed previously to catalyze the oxidation of L-proline to provide L-glutamate semialdehyde (L-GSA) (31) as a precursor for the condensation reaction catalyzed by CarB.…”

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confidence: 60%

Mutational Analysis of the Thienamycin Biosynthetic Gene Cluster from Streptomyces cattleya

Rodríguez

Núñez

Braña

et al. 2011

Antimicrob Agents Chemother

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The generation of non-thienamycin-producing mutants with mutations in the thnL, thnN, thnO, and thnI genes within the thn gene cluster from Streptomyces cattleya and their involvement in thienamycin biosynthesis and regulation were previously reported. Four additional mutations were independently generated in the thnP, thnG, thnR, and thnT genes by insertional inactivation. Only the first two genes were found to play a role in thienamycin biosynthesis, since these mutations negatively or positively affect antibiotic production. A mutation of thnP results in the absence of thienamycin production, whereas a 2-to 3-fold increase in thienamycin production was observed for the thnG mutant. On the other hand, mutations in thnR and thnT showed that although these genes were previously reported to participate in this pathway, they seem to be nonessential for thienamycin biosynthesis, as thienamycin production was not affected in these mutants. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of all available mutants revealed some putative intermediates in the thienamycin biosynthetic pathway. A compound with a mass corresponding to carbapenam-3-carboxylic acid was detected in some of the mutants, suggesting that the assembly of the bicyclic nucleus of thienamycin might proceed in a way analogous to that of the simplest natural carbapenem, 1-carbapen-2-em-3-carboxylic acid biosynthesis. The accumulation of a compound with a mass corresponding to 2,3-dihydrothienamycin in the thnG mutant suggests that it might be the last intermediate in the biosynthetic pathway. These data, together with the establishment of cross-feeding relationships by the cosynthesis analysis of the non-thienamycin-producing mutants, lead to a proposal for some enzymatic steps during thienamycin assembly.

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confidence: 60%

Mutational Analysis of the Thienamycin Biosynthetic Gene Cluster from Streptomyces cattleya

Rodríguez

Núñez

Braña

et al. 2011

Antimicrob Agents Chemother

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“…In the CEA⅐AMP-CPP͞Mg 2ϩ and DGPC⅐AMP͞PP i ͞Mg 2ϩ structures, Tyr-348 is further away, with a hydroxyl oxygen to ␣-nitrogen distance of Ϸ5 Å. Notably, both Tyr-348 and Glu-382 are conserved in CarA, a ␤-LS homolog that catalyzes similar chemistry in carbapenem biosynthesis (29,30). Neither residue is conserved in AS-B, although Glu-382 is replaced with aspartic acid.…”

Section: Discussionmentioning

confidence: 99%

The catalytic cycle of β-lactam synthetase observed by x-ray crystallographic snapshots

Miller

Bachmann

Townsend

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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The catalytic cycle of the ATP͞Mg 2؉ -dependent enzyme ␤-lactam synthetase (␤-LS) from Streptomyces clavuligerus has been observed through a series of x-ray crystallographic snapshots. Chemistry is initiated by the ordered binding of ATP͞Mg 2؉ and N 2 -(carboxyethyl)-L-arginine (CEA) to the apoenzyme. The apo and ATP͞Mg 2؉ structures described here, along with the previously described CEA⅐␣,␤-methyleneadenosine 5 -triphosphate (CEA⅐AMP-CPP)͞Mg 2؉ structure, illuminate changes in active site geometry that favor adenylation. In addition, an acyladenylate intermediate has been trapped. The substrate analog N 2 -(carboxymethyl)-L-arginine (CMA) was adenylated by ATP in the crystal and represents a close structural analog of the previously proposed CEA-adenylate intermediate. Finally, the structure of the ternary product complex deoxyguanidinoproclavaminic acid (DGPC)⅐AMP͞PP i͞Mg 2؉ has been determined. The CMA-AMP͞PP i͞Mg 2؉ and DGPC⅐AMP͞PPi͞Mg 2؉ structures reveal interactions in the active site that facilitate ␤-lactam formation. All of the ATP-bound structures differ from the previously described CEA⅐AMP-CPP͞Mg 2؉ structure in that two Mg 2؉ ions are found in the active sites. These Mg 2؉ ions play critical roles in both the adenylation and ␤-lactamization reactions.

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“…In Pectobacterium carotovora, only three enzymes are required to catalyze formation of the simplest carbapenem, (5R)-carbapenem-3-carboxylate from primary metabolic precursors (13). Formation of carboxymethylproline (t-CMP) 2 the ␤-amino acid precursor of the first formed ␤-lactam ((3S,5S)-carbapenam-3-carboxylate) is catalyzed by a member of the crotonase superfamily, carboxymethylproline synthase (CarB), in a reaction involving malonyl-CoA and L-glutamate semialdehyde (L-GSA) substrates (14,15) (Scheme 1).…”

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confidence: 99%

Structural and Mechanistic Studies on Carboxymethylproline Synthase (CarB), a Unique Member of the Crotonase Superfamily Catalyzing the First Step in Carbapenem Biosynthesis

Sleeman

Sorensen

Batchelar

et al. 2005

Journal of Biological Chemistry

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There are four main subfamilies of clinically used bicyclic ␤-lactam antibiotics comprised of the penicillins, the cephalosporins, the carbapenems, and the oxacephems. Three inhibitors of the serine ␤-lactamases have also found extensive clinical use and although these compounds are themselves bicyclic ␤-lactams, they possess limited antibiotic activity; hence are used in conjunction with a penicillin. With

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Three Unusual Reactions Mediate Carbapenem and Carbapenam Biosynthesis

Cited by 63 publications

References 21 publications

Mutational Analysis of the Thienamycin Biosynthetic Gene Cluster from Streptomyces cattleya

Mutational Analysis of the Thienamycin Biosynthetic Gene Cluster from Streptomyces cattleya

The catalytic cycle of β-lactam synthetase observed by x-ray crystallographic snapshots

Structural and Mechanistic Studies on Carboxymethylproline Synthase (CarB), a Unique Member of the Crotonase Superfamily Catalyzing the First Step in Carbapenem Biosynthesis

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