Structure and Catalysis in the Escherichia coli Hotdog-fold Thioesterase Paralogs YdiI and YbdB

The hotdog-fold superfamily is a functionally diverse family of evolutionarily related enzymes, which share a common α + β-fold. The founding member of this superfamily is the Escherichia coli β-hydroxydecanoyl-holo acyl carrier protein (ACP) dehydrase/isomerase.a5 Smith and co-workers coined the name “hotdog-fold” to represent the observed tertiary structure consisting of a 5-turn α-helix cradled by a curved, 7-stranded antiparallel β-sheet. The functional unit was reported as a dimer, with the subunit interface joining the two sheets into a continuous 14-stranded β-sheet. The two active sites are located at opposite ends of the interfaced sheets. Soon after the report of the dehydratase/isomerase, the hotdog-fold was observed in the 4-hydroxybenzoyl-coenzyme A thioesterase from Pseudomonas sp. strain CBS3 (Ps 4-HB-CoA TE)1 and shortly thereafter in the 4-HB-CoA thioesterase from Arthrobacter special strain AU (Ar 4-HB-CoA TE)3 (Figure ​(Figure1A1A and B). Both thioesterases catalyze the final step of the 4-chlorobenzoate-degradation pathway.6,7 Surprisingly, the two thioesterases share little sequence identity (<25%), and although the tertiary structures are alike, the arrangements of the dimers, which form the respective (biological unit) tetramers, differ. Specifically, the dimers of the Ar 4-HB-CoA TE associate β-sheet-to-β-sheet, with the active sites directed to solvent, whereas in the Ps 4-HB-CoA TE structure the dimers are rotated 180° such that the active sites are directed to the dimer–dimer interface.1−3 Figure 1 (A) Tetrameric structure (dimer of dimers) of the hotdog-fold superfamily clade AA member 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas sp. strain CBS3 represented in Pymol.2 The two structural representations are related by a 90° vertical rotation. ... Most relevant to the catalytic mechanism is the fact that even though the secondary structural elements that form the active sites of the two thioesterases are highly conserved, the constellation of catalytic residues is dissimilar as is the spatial location of the essential catalytic carboxylate residue.2,3 Based on the noted structural differences, the Ar 4-HB-CoA TE and Ps 4-HB-CoA TE are viewed as prototypes of two evolutionarily distinct clades of thioesterases within the hotdog-fold superfamily.b Notably, the common fold that links these two structural clades dictates that, for all members, substrate recognition is directed at holoACP or CoA-based thioester metabolites. The thioesterases constitute a large subfamily within the hotdog-fold superfamily. The biological function of the thioesterase is defined by the identity of its in vivo substrate(s) (i.e., physiological substrate(s)) in combination with the biochemical context, as determined by protein partners and/or elements of regulation. When subjected to an in vitro activity screen, the typical hotdog-fold thioesterase displays hydrolytic activities toward a range of thioesters that extend well beyond the physiological substrate(s).8−12 Whereas this lack of substrate specificity might, in earlier times, have been interpreted as evidence for an “under-evolved” enzyme (befitting the role of “scavenger”), it is now well recognized that substrate promiscuity is a key trait of highly evolvable enzymes (for review, see ref (13)). We posit that this trait is inherent in the simplicity and plasticity of the architecture of the hotdog-fold. Presently, we have carried out a case study of two paralogs, namely, YdiI and YbdB from E. coli. These thioesterases are related by 59% sequence identity and are distinguished by very diverse biological functions as detailed in the preceding companion paper14 and depicted in Scheme 1. Briefly, in E. coli, YdiI participates in menaquinone biosynthesis as a catalyst for the hydrolysis of the pathway intermediate, 1,4-dihydroxynapthoyl-CoA.14−16 YbdB, on the other hand, assists in the biosynthesis of the siderophore enterobactin through catalyzed hydrolysis of aberrant aroyl-holoEntB thioesters that, as dead-end pathway intermediates, act to block enterobactin formation.17−19 Scheme 1 Biological Functions of E. coli YdiI and YbdB Herein, the structural determinants for substrate recognition and catalysis in these two paralogs are reported, and analyzed, within the context of evolution of hotdog-fold thioesterase function. In addition, because YdiI and YbdB are members of the same family clade as Ar 4-HB-CoA TE (Figure ​(Figure1B1B and C),3 a comparison of the relevant elements of structure and mechanism with those of this evolutionarily more distant relative is made, to identify those elements, which have been retained despite extensive sequence divergence. The findings are interpreted to suggest that a phosphopantethiene binding site is conserved for targeting CoA and holoACP-based thioester metabolites, and that the location of the ACP or adenosine 3′,5′-diphosphate binding site on the surface of the thioesterase serves to promote promiscuity toward these two classes of thioesters. Furthermore, we infer that the divergence in the alkyl/aryl binding site sequence is important for the evolution of new biological function. Finally, we propose that the roles played by the conserved catalytic residues in thioester hydrolysis change in the process of adaptation of the thioesterase to its new physiological substrate.