Definition of the Common and Divergent Steps in Carbapenem β‐Lactam Antibiotic Biosynthesis

Abstract

Approximately 50 naturally occurring carbapenem β-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.

Consecutive radical S -adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis

“…These natural products have been isolated from actinomyces, whereas the lone "simple" carbapenem, carbapenem-3-carboxylic acid 3, is known from an enterobacterium, Pectobacterium carotovorum (1). Despite the evolutionary distance between the producers, the first two biosynthetic steps to each are shared (2). Thus, CarB encoded in the latter and ThnE from Streptomyces cattleya, and, correspondingly, CarA and ThnM give (3S,5S)-carbapenam 2 ( Fig.…”

Autoproteolytic Activation of ThnT Results in Structural Reorganization Necessary for Substrate Binding and Catalysis

“…12, 35, 36 Although the data presented here do not provide a ready explanation for the inability of ThnT to cleave phosphopanetheine, the catalytic activity of ThnT is clear. Formation of the Michaelis complex places the carbonyl of the scissile amide into the oxyanion hole (Fig.…”

Engineering the synthetic potential of β-lactam synthetase and the importance of catalytic loop dynamics

“…21 In the case of CPS from simple carbapenem biosynthesis, this histidine (also His447) may interact with either or both Tyr467 and Glu322, which occupy the same region as Thr453 is in βLS. In ThnM, the CPS homolog active in thienamycin biosynthesis, 22 Thr453 is replaced by Ser407, which presumably would interact with His403 in that enzyme. On the other hand, Val446 is substituted by an isoleucine in a βLS paralog 23 and in both CPS 16 and ThnM and their homologs 24 , so mutation here to another nonpolar residue must not have a great effect on the wild-type reaction, as confirmed by the behavior of the V446A mutation.…”