Divergence of Function in the Hot Dog Fold Enzyme Superfamily: The Bacterial Thioesterase YciA † Zhihao Zhuang, ‡ Feng Song, ‡ Hong Zhao, ‡ Ling Li, ‡ Jian Cao, ‡ Edward Eisenstein, § Osnat Herzberg, § and Debra Dunaway-Mariano* ,‡ Department of Chemistry, UniVersity of New Mexico, Albuquerque, New Mexico 81713, and Center for AdVanced Research in Biotechnology, UniVersity of Maryland Biotechnology Institute, RockVille, Maryland 20850 ReceiVed NoVember 26, 2007; ReVised Manuscript ReceiVed January 9, 2008 ABSTRACT: Thioesters play a central role in the cells where they participate in metabolism, membrane synthesis, signal transduction, and gene regulation. Thioesters are converted to the thiol and carboxylic acid components by thioesterase-catalyzed hydrolysis. Here we examine the biochemical and biological function of the hot dog fold thioesterase YciA (EcYciA) from Escherichia coli and its close sequence homologue HI0827 from Haemophilus influenzae (HiYciA). The quaternary structure of HiYciA was determined, using equilibrium sedimentation techniques, to be a homohexamer. Mass spectral and 31 P NMR analysis of purified HiYciA revealed a bound CoA ligand. Kinetic analyses showed that CoA is a strong feedback inhibitor. YciA thioesterase activity toward acyl-CoA substrates was determined using steady-state kinetic methods. The k cat and k cat /K m values obtained reveal a striking combination of high catalytic efficiency and low substrate specificity. The substrate activity of propionyl-s-N-acetylcysteine was found to be negligible and that of n-butyryl-pantetheinephosphate low, and therefore, it is evident YciA does not target acylated ACPs or other acylated proteins as substrates. The results from bioinformatic analysis of the biological distribution and genome contexts of yciAs are reported. We conclude that YciA is responsible for the efficient, “seemingly” indiscriminant, CoA-regulated hydrolysis of cellular acyl- CoA thioesters in a wide range of bacteria and hypothesize that this activity may support membrane biogenesis. Thioesters play a central role in the cells where they participate in metabolism, membrane synthesis, signal trans- duction, and gene regulation (1). The carboxylic acid components of biological thioesters are metabolites of varied size, shape, and polarity. They are converted to thioesters by ligases for the purpose of solubility, transport, signaling, or activation for reaction in biosynthetic or biodegradation pathways. The naturally occurring thiols include coenzyme A (CoA), 1 glutathione, the pantetheine unit of the holoacyl carrier protein (ACP), and the cysteine residue of a protein. Thioesters are reverted to the thiol and carboxylic acid components by hydrolysis catalyzed by thioesterases. Thioesterases have primarily evolved within the R/-fold hydrolase enzyme superfamily (2) and the hot dog fold enzyme superfamily (3). The conserved fold of the hot dog fold superfamily proteins consists of a five-stranded anti- parallel -sheet wrapped around an elongated R-helix. This topology is reminiscent of a hot dog “bun” wrapping around a “sausage”, hence the name hot dog fold (4). Following the discovery of the first hot dog fold thioesterase (viz., the 4-hydroxybenzoyl-CoA thioesterase from Pseudomo- nas sp. strain CBS3) (5), we embarked on a long-term study of the diversification of function within the hot dog fold thioesterase family (6–14). During the course of this work, we have discovered that (i) the hot dog fold is conserved despite the low degree of amino acid sequence conservation within the family, 2 (ii) divergent evolution within this family is based on two rather than one catalytic scaffold (6), (iii) there is no conservation of a set of core catalytic residues and, instead, one carboxylate residue [Asp or Glu located at either of two positions of the catalytic scaffold (6, 12)] operates within a variety of sequence contexts, and (iv) the absence of well-defined, desolvated substrate binding pockets is responsible for low substrate specificity (13). These family features combine in such a way to severely impede a sequence-based, and even a three-dimensional structure- based, approach to function assignment. It is for this reason that we have focused our attention on a set of hot dog thioesterases that are produced by the well-characterized organism Escherichia coli so that we may use gene context, as well as the knowledge of metabolic pathways and known † This work was supported by National Institutes of Health Grants P01 GM57890 (O.H.) and R01 GM28688 (D.D.-M.). * To whom correspondence should be addressed: Department of Chemistry and Chemical Biology, University of New Mexico, Albu- querque, NM 81713. Telephone: (505) 277-3383. Fax: (505) 277-6202. E-mail: dd39@unm.edu. ‡ University of New Mexico. § University of Maryland Biotechnology Institute. 1 Abbreviations: DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); CoA, coenzyme A; DTT, dithiothreitol; IPTG, isopropyl -D-thiogalactopy- ranoside; PMSF, phenylmethanesulfonyl fluoride; K + Hepes, potassium salt of N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonate; ACP, acyl carrier protein; 4-HBA-CoA, 4-hydroxybenzoyl-coenzyme A; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2 For example, the pairwise sequence alignment of E. coli YciA with YbgC (14% identical), YbaW (8% identical), YbdB (17% identical), YdiI (15% identical), and PaaI (13% identical) reveals no significant sequence homology. Biochemistry 2008, 47, 2789–2796 2789 10.1021/bi702334h CCC: $40.75  2008 American Chemical Society Published on Web 02/02/2008