Non-Heme Iron Oxygenases Generate Natural Structural Diversity in Carbapenem Antibiotics

Abstract

Carbapenems are a clinically important antibiotic family. More than 50 naturally occurring carbapenam/ems are known and are distinguished primarily by their C-2/C-6 side chains where many are only differentiated by the oxidation states of these substituents. With a limited palette of variations the carbapenem family comprises a natural combinatorial library, and C-2/C-6 oxidation is associated with increased efficacy. We demonstrate that ThnG and ThnQ encoded by the thienamycin gene cluster in Streptomyces cattleya oxidize the C-2 and C-6 moieties of carbapenems, respectively. ThnQ stereospecifically hydroxylates PS-5 (5) giving N-acetyl thienamycin (2). ThnG catalyzes sequential desaturation and sulfoxidation of PS-5 (5), giving PS-7 (7) and its sulfoxide (9). The enzymes are relatively substrate selective but are proposed to give rise to the oxidative diversity of carbapenems produced by S. cattleya, and orthologues likely function similarly in allied streptomyces. Elucidating the roles of ThnG and ThnQ will focus further investigations of carbapenem antibiotic biosynthesis.

Mutational Analysis of the Thienamycin Biosynthetic Gene Cluster from Streptomyces cattleya

“…This role for ThnQ indirectly suggests that the early oxidation step converting the carbapenam ring to carbapenem might be carried out by ThnQ earlier on in the pathway. In addition, ThnG was recently found to give rise to oxidative carbapenem diversity being able to hydroxylate the C-6 carbapenem moiety in vitro (2). Third, the 2.5-fold increase in thienamycin production observed for the thnG deletion mutant, in comparison with the S. cattleya wild-type strain, might be explained by a putative lack of expression of the downstream thnF gene as a consequence of the mutation.…”

“…As mentioned above, the encoding genes display a different regulation pattern, and the thnQ monocistronic transcripts were found to be five times more abundant than the monocistronic transcripts for thnG (27). The function of both enzymes in the thienamycin biosynthetic pathway has been the subject of multiple hypotheses (2,13,25,30). A prominent feature of the role of the 2-oxoglutarate-dependent oxygenases in ␤-lactam biosynthesis pathways is their ability to catalyze more than one reaction (15).…”

“…In addition, ThnF was found to be most likely responsible for the formation of the S. cattleya cometabolite N-acetylthienamycin in the presence of acetyl-CoA (12). Recombinant versions of ThnG and ThnQ were recently shown to generate in vitro structural diversity in carbapenem antibiotics (2).…”

Oxidative Tailoring Reactions Catalyzed by Nonheme Iron-Dependent Enzymes

“…The C-6 substituent (an ethyl group in thienamycin) can be a methyl, ethyl, or isopropyl group and can be saturated, unsaturated, hydroxylated, or sulfated. The C-2 cysteamine, or more rarely pantetheine, substituent can be N-acylated, N-propionylated, desaturated, or oxidized to a sulfoxide, or even cleaved and oxidized to a sulfonic acid (Bodner, Phelan, Freeman, Li, & Townsend, 2010). Several carbapenems are clinically used antibiotics.…”

“…Some carbapenems with oxidized C-2/C-6 substituents have increased antibiotic activity or higher resistance to b-lactamases. As a consequence, investigation of oxidations of the carbapenem C-2/C-6 substituent has been of particular interest (Bodner et al, 2010).…”