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...

Heparanase (HPA) is a critical enzyme involved in the remodeling of the extracellular matrix (ECM), and its elevated expression has been linked with diseases such as various types of cancer...

The enzyme 2-keto-3-deoxy-9-O-phosphonononic acid phosphatase (KDN9P phosphatase) functions in the pathway for the production of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, a sialic acid that is important for the survival of commensal bacteria in the human intestine. The enzyme is a member of the haloalkanoate dehalogenase superfamily and represents a good model for the active-site protonation state of family members. Crystals of approximate dimensions 1.5 × 1.0 × 1.0 mm were obtained in space group P2(1)2(1)2, with unit-cell parameters a = 83.1, b = 108.9, c = 75.7 Å. A complete neutron data set was collected from a medium-sized H/D-exchanged crystal at BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany in 18 d. Initial refinement to 2.3 Å resolution using only neutron data showed significant density for catalytically important residues.

Heparanase is a critical enzyme involved in the remodeling of the extracellular matrix (ECM), and its elevated expression has been linked with diseases such as cancer and inflammation. The detection of heparanase enzymatic activity holds tremendous value in the study of the cellular microenvironment, and search of molecular therapeutics targeting heparanase, however, assays developed for this enzyme so far have suffered prohibitive drawbacks. Here we present an ultrasensitive fluorogenic small-molecule probe for heparanase enzymatic activity. The probe exhibits a 756-fold fluorescence turn-on response in the presence of human heparanase, allowing one-step detection of heparanase activity in real-time with a picomolar detection limit. The high sensitivity and robustness of the probe are exemplified in a high-throughput screening assay for heparanase inhibitors.Heparanase, an endo-β-glucuronidase of the glycoside hydrolase 79 (GH79) family 1,2 , is responsible for the cleavage of heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPG) 3 . These protein-polysaccharide conjugated macromolecules, abundantly expressed in the extracellular matrix (ECM), play an essential structural role in maintaining the ECM integrity.Moreover, the HS side chains bind to an array of biological effector molecules, such as growth factors, chemokines, and cytokines, thereby serving as their reservoir that can liberate the desired signaling molecules when needed.

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