YbtT is a low-specificity type II thioesterase that maintains production of the metallophore yersiniabactin in pathogenic enterobacteria

Abstract

Edited by Ruma BanerjeeClinical isolates of Yersinia, Klebsiella, and Escherichia coli frequently secrete the small molecule metallophore yersiniabactin (Ybt), which passivates and scavenges transition metals during human infections. YbtT is encoded within the Ybt biosynthetic operon and is critical for full Ybt production in bacteria. However, its biosynthetic function has been unclear because it is not essential for Ybt production by the in vitro reconstituted nonribosomal peptide synthetase/polyketide synthase (NRPS/ PKS) pathway. Here, we report the structural and biochemical characterization of YbtT. YbtT structures at 1.4 -1.9 Å resolution possess a serine hydrolase catalytic triad and an associated substrate chamber with features similar to those previously reported for low-specificity type II thioesterases (TEIIs). We found that YbtT interacts with the two major Ybt biosynthetic proteins, HMWP1 (high-molecular-weight protein 1) and HMWP2 (high-molecular-weight protein 2), and hydrolyzes a variety of aromatic and acyl groups from their phosphopantetheinylated carrier protein domains. In vivo YbtT titration in uropathogenic E. coli revealed a distinct optimum for Ybt production consistent with a tradeoff between clearing both stalled inhibitory intermediates and productive Ybt precursors from HMWP1 and HMWP2. These results are consistent with a 6 The abbreviations used are:

Leveraging copper import by yersiniabactin siderophore system for targeted PET imaging of bacteria

“…coli UTI89 ( 6 ), E . coli Nissle WT, and Klebsiella pneumoniae ( 9 , 10 ) compared with those infected with control bacteria that do not express a functional FyuA transporter protein. Staphylococcus aureus was used as control as it does not possess the YbT transport machinery.…”

Structure, function, and regulation of thioesterases

“…YbtT is a thioesterase encoded within the yersiniabactin biosynthetic operon of Yersinia spp. and uropathogenic E. coli, and is thought to remove aberrant yersiniabactin precursors from carrier proteins, that would otherwise stall or inhibit yersiniabactin sysnethesis [189]. YbtT and RifR have been suggested to behave as low-specificity editing thioesterases, meaning they would display greater substrate promiscuity in regards to acyl chain length than others thioesterases, such as RedJ.…”

“…YbtT and RifR have been suggested to behave as low-specificity editing thioesterases, meaning they would display greater substrate promiscuity in regards to acyl chain length than others thioesterases, such as RedJ. This appears to be at least partly due to the composition of residues within the flexible lid region of these enzymes, with RedJ having a greater composition of hydrophobic residues in the lid region compared to RifR and YbtT, in which the hydrophobic residues are largely replaced by polar residues [185,189].…”

Radiolabelled Bacterial Metallophores as Targeted PET Imaging Contrast Agents for Accurate Identification of Bacteria and Outer Membrane Vesiclesin vivo

“…Besides E. coli UTI89, probiotic E. coli Nissle and pathogenic Klebsiella pneumoniae also transport metals using YbT via the FyuA receptor [26][27][28] . We next reasoned that if radiometallabelled YbT is truly selective for FyuA only, then our probes should be able to accumulate in Nissle and in K. pneumoniae as well.…”