The Mechanisms of Human Hotdog-fold Thioesterase 2 (hTHEM2) Substrate Recognition and Catalysis Illuminated by a Structure and Function Based Analysis^,

Abstract

The focus of this paper is the hotdog-fold thioesterase THEM2 from human (hTHEM2; Swiss-Prot entry Q9NPJ3). In an earlier communication (Cheng, Z., Song, F., Shan, X., Wei, Z., Wang, Y., Dunaway-Mariano, D., and Gong, W. (2006) Crystal structure of human thioesterase superfamily member 2, Biochem Biophys Res Commun 349, 172-177.) we reported the apo crystal structure of hTHEM2. Herein, we report the results of an extensive hTHEM2 substrate screen, the structure determination of hTHEM2 complexed with the inert substrate analog undecan-2-one-CoA (in which O=C-CH2-S substitutes for O=C-S) and the kinetic analysis of active site mutants. The work described in this paper represents the first reported structure-function based analysis of a human hotdog-fold thioesterase. The catalytic mechanism proposed involves the Asp65/Ser83 assisted attack of a water molecule at the Gly57/Asn50 polarized thioester C=O and the Asn50 assisted departure of the thiolate leaving group. Thioesterase activity was observed with acyl-CoAs but not with the human acyl-ACP or with an acyl-Cys peptide. The medium-to-long chain fatty acyl-CoAs displayed the smallest Km values. The substrate specificity profile was analyzed within the context of the liganded enzyme to define the structural determinants of substrate recognition. Based on the results of this structure-function analysis we hypothesize that the physiological role of hHTEM2 involves catalysis of the hydrolysis of cytosolic medium-to-long chain acyl-CoA thioesters.

Functional characterization of thioesterase superfamily member 1/Acyl-CoA thioesterase 11: implications for metabolic regulation

“…This suggests 25 µM palmitoylCoA, which we used in our oligomerization studies, should fall within the biological range. Such intracellular fatty acyl-CoA concentrations would also be in keeping with the K m values we observed for Them1 and with those reported for other Acots ( 8,22,23,28 ). However, fatty acyl-CoAs may bind to proteins and cell membranes, and this would presumably reduce their accessibility to Acots.…”

Acyl Coenzyme A Thioesterase Them5/Acot15 Is Involved in Cardiolipin Remodeling and Fatty Liver Development

“…S2D in the supplemental material). These residues are proposed to bind and hydrolyze the thioester moiety of the acyl-CoA substrate, as described for a number of other Hotdog thioesterases (8,26). They are located in the middle of a long L-shaped channel that is limited in length by a stretch of residues from the N-terminal part (residues 74 to 79 in ⌬36Them4 and residues 83 to 88 in ⌬34Them5), which likely defines the length of the CoA-coupled fatty acids accepted for turnover.…”

“…However, in both KO and HET mitochondria, the common effect of Them5 on cardiolipin was altered ratios of major species of CL and MLCL (see Fig. S4C in the supplemental material), analogous to the phenotypic changes in Barth syndrome (8). Subsequently, we focused on characterization of the complete Them5 knockout.…”

“…Of the eight Hotdog-fold proteins identified thus far in mammals, only a few participate in biological processes involving hydrolysis of acylCoAs (5). These are cytoplasmic Acot7 involved in eicosanoid metabolism, Acot11 and Acot12, which have a START domain, and cytoplasmic/mitochondrial Them2, which is induced by peroxisome proliferator-activated receptor ␣ (PPAR␣) and has been recently shown to regulate hepatic metabolism (5,8,16,18). The mammalian Hotdog thioesterases identified thus far have tetrameric or hexameric arrangements (26).…”

Thioesterase Superfamily Member 2 (Them2) and Phosphatidylcholine Transfer Protein (PC-TP) Interact To Promote Fatty Acid Oxidation and Control Glucose Utilization

“…hioesterase superfamily member 2 (Them2), a member of the acyl coenzyme A (CoA) thioesterase (Acot) family, catalyzes the hydrolysis of long-chain fatty acyl-CoA esters to free fatty acids plus CoA (1,2). Them2 is enriched in liver and oxidative tissues, including brown adipose (3)(4)(5).…”