Divergence of Function in the Hot Dog Fold Enzyme Superfamily: The Bacterial Thioesterase YciA

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.

Enzyme Promiscuity: Engine of Evolutionary Innovation

“…The broad observed substrate range (k cat /K m ϭ 1.1 ϫ 10 7 and 1.4 ϫ 10 6 M Ϫ1 s Ϫ1 for isobutyryl-CoA and lauroyl-CoA, respectively), together with the gene context, suggests a role for YciA and its orthologs in recycling CoA and balancing fatty acyl-CoA pools for membrane remodeling. As in the thioesterase EntH, subsequent YciA structure determination revealed that substrate recognition is directed at the thioester pantetheine moiety and not the acyl moiety (37), thus accounting for the observed promiscuity, as well as its tight regulation by strong CoA feedback inhibition (24) (Fig. 1).…”

“…In the thioesterases displaying a very restricted substrate range, the alkyl/aryl-binding site is an enclosed pocket (for example, fluoroacetyl-CoA (57, 58) and 4-hydroxybenzoyl-CoA thioesterases (54,55,59)). In contrast, for thioesterases displaying a modest (60) or expansive substrate range, like hTHEM2 and hTHEM4 (61,62) and E. coli YciA (24,37), the pocket is partially open or leads to solvent, respectively. Notably, the fact that catalytic efficiency and substrate ambiguity can co-exist is exemplified by the thioesterase YciA where k cat /K m values range from 3 ϫ 10 5 to 8 ϫ 10…”

“…Substrate specificity profiles have been reported for numerous enzyme classes including phosphatases (19 -23), thioesterases (24 -26), peptide racemases (27), and lipases/phospholipases (28). The substrate structures can be surprisingly varied; for instance, single enzymes can utilize long and short-chain acyl-CoA substrates (24), apolar and anionic lipids (28), or various length phosphosugars and nucleotides (20).…”

“…Ϫ1 s Ϫ1 for CoA thioester substrates as diverse as acetyl-CoA, phenylacetylCoA, and myristoyl-CoA (24).…”

Novel functions of acyl-CoA thioesterases and acyltransferases as auxiliary enzymes in peroxisomal lipid metabolism

“…This is in fact one of the few crystal structures currently available for homologues of the mammalian ACOT enzymes, however work is currently underway to crystallize several of these mammalian proteins. In E. coli however, there are seven different hot dog thioesterases encoded by the E. coli genome that have a known X-ray structure, the biological function is only known in the case of one thioesterase [41]. …”