Enoyl-CoA hydratase (EC 4.2.1.17) (ECH) [1] and D3,D2-enoyl-CoA isomerase (EC 5.3.3.8) (ECI) [2] are two enzymes involved in fatty acid metabolism. ECH catalyzes the hydratation of 2-trans-enoyl-CoA into 3-hydroxyacyl-CoA and ECI shifts the 3- double bond of the intermediates of unsaturated fatty acid oxidation to the 2-trans position.

Most eukaryotic cells have two fatty-acid β-oxidation systems, one located in mitochondria and the other in peroxisomes. In mitochondria, ECH and ECI are separate yet structurally related monofunctional enzymes. Peroxisomes contain a trifunctional enzyme [3] consisting of an N-terminal domain that bears both ECH and ECI activity, and a C-terminal domain responsible for 3-hydroxyacyl-CoA dehydrogenase (HCDH) activity.

In Escherichia coli (gene fadB) and Pseudomonas fragi (gene faoA), ECH and ECI are also part of a multifunctional enzyme which contains both a HCDH and a 3-hydroxybutyryl-CoA epimerase domain [4].

A number of other proteins have been found to be evolutionary related to the ECH/ECI enzymes or domains:

• 3-hydroxbutyryl-coa dehydratase (EC 4.2.1.55) (crotonase), a bacterial enzyme involved in the butyrate/butanol-producing pathway.
• Naphthoate synthase (EC 4.1.3.36) (DHNA synthase) (gene menB) [5], a bacterial enzyme involved in the biosynthesis of menaquinone (vitamin K2). DHNA synthase converts O-succinyl-benzoyl-CoA (OSB-CoA) to 1,4-dihydroxy-2- naphthoic acid (DHNA).
• 4-chlorobenzoate dehalogenase (EC 3.8.1.6) [6], a Pseudomonas enzyme which catalyzes the conversion of 4-chlorobenzoate-CoA to 4-hydroxybenzoate-CoA.
• A Rhodobacter capsulatus protein of unknown function (ORF257) [7].
• Bacillus subtilis putative polyketide biosynthesis proteins pksH and pksI.
• Escherichia coli carnitine racemase (gene caiD) [8].
• Escherichia coli hypothetical protein ygfG.
• Yeast hypothetical protein YDR036c.

As a signature pattern for these enzymes, we selected a conserved region rich in glycine and hydrophobic residues.