In Vitro Kinetic Analysis of Substrate Specificity in Enterobactin Biosynthetic Lower Pathway Enzymes Provides Insight into the Biochemical Function of the Hot Dog-Fold Thioesterase EntH

Abstract

The Escherichia coli siderophore enterobactin is assembled from 2,3-dihydroxybenzoate (2,3-DHB) and L-serine by the nonribosomal peptide synthetases EntB and EntF. The processive thiol-template strategy used can be sabotaged by EntB misacylation. Through in vitro kinetic analysis we demonstrate two potential routes to EntB misacylation and provide evidence for two mechanisms by which the hotdog-fold thioesterase EntH can potentially prevent or reverse EntB misacylation.The siderophore enterobactin is synthesized by Escherichia coli to function in the harvesting of exogenous iron (1). Because pathogenic strains must sequester iron from the human host, the enterobactin biosynthetic enzymes are ideal targets for the development of novel antibiotics (2). Enterobactin biosynthesis proceeds via an upper chemical pathway that forms 2,3-dihydroxybenzoate (2,3-DHB) from chorismate and a lower, iterative pathway that assembles enterobactin from L-serine and 2,3-DHB. The lower pathway (Fig. 1), which is the focus of our work, employs the two nonribosomal peptide synthetases (NRPSs) EntB and EntF (3). The phosphopantetheinyl transferase (PPTase) "EntD" catalyzes the transfer of pantetheinephosphate from CoA to the Ser residue of the 2,3-DHB-carrier domain of EntB and of the peptidyl-carrier domain of EntF to generate holo-EntB and holo-EntF, respectively. EntE catalyzes the adenylation of 2,3-DHB and the subsequent aroyl transfer to the pantetheine thiol of holo-EntB. The adenylation domain of holo-EntF catalyzes the adenylation of L-Ser and subsequent acyl-transfer to the pantetheine thiol carrier domain of holo-EntF. The EntF condensation domain then catalyzes the transfer of the 2,3-DHB unit to the amine group of the EntF tethered L-Ser. The 2,3-DHB-Ser unit is then transferred to the active site Ser of the EntF cyclizing thioesterase domain, thereby freeing the pantetheine thiol for acquisition of a second 2,3-DHB-Ser-DHB unit. It in turn is transferred to the Ser moiety side chain of 2,3-DHB-Ser tethered to the TE domain. Following the addition of a third 2,3-DHB-Ser unit, the chain is cyclized by the thioesterase domain to form enterobactin.The genes encoding the enterobactin biosynthetic enzymes are clustered within cotranscriptional units, one of which includes EntC and EntA of the upper pathway and EntE, EntB and EntH (also known as ybdB) of the lower pathway. EntH is a member of the hotdogfold thioesterase family (4). The biosynthetic pathways leading to NRPs in other bacteria, possess stand alone thioesterases from the α/β-hydrolase-fold family. Previous studies have ‡ This work was supported by NIH grant GM28688. provided evidence for the house-keeping role played by these enzymes, involving removal of non-native acyl units from the pantetheine thiols of misacylated NRPSs (5)- (7). Bouveret and coworkers (8) used in vivo two-hybrid and in vitro co-purification techniques to demonstrate interaction between EntH and holo-EntB. These investigators proposed a proofreading role for EntH in which holo-EntB acy...

Structure, Activity, and Substrate Selectivity of the Orf6 Thioesterase from Photobacterium profundum

“…However, this seems unlikely because most hotdog TEs are specific for the hydrolysis of CoA thioesters (14,28). For instance, in vitro assays showed that the human hotdog fold TE (hTHEM2) is unable to hydrolyze ACP-loaded thioesters, although it has a broad tolerance for many CoA thioesters (14).…”

“…For instance, in vitro assays showed that the human hotdog fold TE (hTHEM2) is unable to hydrolyze ACP-loaded thioesters, although it has a broad tolerance for many CoA thioesters (14). Similarly, the EntH hotdog TE from the enterobactin biosynthetic pathway of E. coli shows no higher selectivity for substrate analogs loaded onto its cognate ACP than for substrates loaded onto CoA, indicating that the EntH TE recognizes the pantetheinyl portion of the substrates with few or no contact with the ACP (28).…”

“…EntH, the hotdog TE involved in the final release of the E. coli natural product enterobactin, is considerably more active than Orf6. Its specific activity ranges from 17 milliunits/mg against acetyl-CoA up to 8400 milliunits/mg against 3-hydroxyphenylacetyl-CoA: an ϳ500-fold preference for aromatic CoA thioesters over acetyl-CoA (28). Similarly, the phenyl acetate-specific TE PaaI from Azoarcus evansii has an ϳ1600-fold preference for 3-hydroxyphenylacetyl-CoA over the shortchain crotonyl-CoA with a range from 17-30,000 milliunits/mg (29).…”

Structure and Catalytic Mechanism of the Thioesterase CalE7 in Enediyne Biosynthesis

“…On the other hand, hotdog fold thioesterases are mainly known to utilize acyl-CoA as substrates, with only a few known examples that act on peptidyl carrier protein or ACP-tethered acyl substrates (23,41,42), including the EntH protein (or YbdB) involved in the biosynthesis of the non-ribosomal peptide-derived enterobactin (41,43,44). Hence, CalE7 appears to represent an uncommon example where a hotdog fold thioesterase was recruited for polyketide synthesis.…”

Enzyme Promiscuity: Engine of Evolutionary Innovation

“…The bifunctional EntE catalyzes the adenylation of 2,3-DHB and the subsequent aroyl transfer to the pantetheine thiol of holo-EntB. Substrate ambiguity of EntD and EntE can derail enterobactin biosynthesis by leading to the formation of misacylated holo-EntB by two means: 1) when CoA is limited, endogenous acyl-CoA may substitute as a substrate for EntD (33), and 2) if 2,3-DHB is limited, a carboxylate metabolite may substitute as a substrate for EntE (34) (Fig. 1).…”