Substrate Report for: Palmitoyl-CoA

Esterases are one of the most common enzymes and are involved in diverse cellular functions. ybfF protein from Escherichia coli (Ec_ybfF) belongs to the esterase family for the large substrates, palmitoyl coenzyme A and malonyl coenzyme A, which are important cellular intermediates for energy conversion and biomolecular synthesis. To obtain molecular information on ybfF esterase, which is found in a wide range of microorganisms, we elucidated the crystal structures of Ec_ybfF in complexes with small molecules at resolutions of 1.1 and 1.68 A, respectively. The structure of Ec_ybfF is composed of a globular alpha/beta hydrolase domain with a three-helical bundle cap, which is linked by a kinked helix to the alpha/beta hydrolase domain. It contains a catalytic tetrad of Ser-His-Asp-Ser with the first Ser acting as a nucleophile. The unique spatial arrangement and orientation of the helical cap with respect to the alpha/beta hydrolase domain form a substrate-binding crevice for large substrates. The helical cap is also directly involved in catalysis by providing a substrate anchor, viz., the conserved residues of Arg123 and Tyr208. The high-resolution structure of Ec_ybfF shows that the inserted helical bundle structure and its spatial orientation with respect to the alpha/beta hydrolase domain are critical for creating a large inner space and constituting a specific active site, thereby providing the broad substrate spectrum toward large biomolecules.

The maintenance of cellular levels of free fatty acids and acyl-CoAs, the activated form of free fatty acids, is extremely important, as imbalances in lipid metabolism have serious consequences for human health. Acyl-coenzyme A (CoA) thioesterases (ACOTs) hydrolyze acyl-CoAs to the free fatty acid and CoASH, and thereby have the potential to regulate intracellular levels of these compounds. We previously identified and characterized a mouse ACOT gene cluster comprised of six genes that apparently arose by gene duplications encoding acyl-CoA thioesterases with localizations in cytosol (ACOT1), mitochondria (ACOT2), and peroxisomes (ACOT3-6). However, the corresponding human gene cluster contains only three genes (ACOT1, ACOT2, and ACOT4) coding for full-length thioesterase proteins, of which only one is peroxisomal (ACOT4). We therefore set out to characterize the human genes, and we show here that the human ACOT4 protein catalyzes the activities of three mouse peroxisomal ACOTs (ACOT3, 4, and 5), being active on succinyl-CoA and medium to long chain acyl-CoAs, while ACOT1 and ACOT2 carry out similar functions to the corresponding mouse genes. These data strongly suggest that the human ACOT4 gene has acquired the functions of three mouse genes by a functional convergent evolution that also provides an explanation for the unexpectedly low number of human genes.

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with "known" function are mis-annotated. Several global approaches are routinely employed to predict function, including sophisticated sequence analysis, gene expression, protein interaction, and protein structure. In the first coupling of genomics and enzymology, Phizicky and colleagues undertook a screen for specific enzymes using large pools of partially purified proteins and specific enzymatic assays. Here we present an overview of the further developments of this approach, which involve the use of general enzymatic assays to screen individually purified proteins for enzymatic activity. The assays have relaxed substrate specificity and are designed to identify the subclass or sub-subclasses of enzymes (phosphatase, phosphodiesterase/nuclease, protease, esterase, dehydrogenase, and oxidase) to which the unknown protein belongs. Further biochemical characterization of proteins can be facilitated by the application of secondary screens with natural substrates (substrate profiling). We demonstrate here the feasibility and merits of this approach for hydrolases and oxidoreductases, two very broad and important classes of enzymes. Application of general enzymatic screens and substrate profiling can greatly speed up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches.