Thioester chemistry is exploited in Nature by many CoA-dependent enzymes. However, the covalent nature of CoA attachment largely prevents the use of these enzymes in many applications. Replacing the CoA moiety with simpler, truncated fragments, such as its pantetheine (PAN) moiety, is also hampered by the lack of understanding of the function of the CoA moiety in enzymatic conversions. Herein, we describe the utilization of the enzyme (2E)-enoyl-CoA hydratase (ECH) using PAN thioesters and an activator, 3′,5′-ADP (PAP). ECH catalyzes the hydration of the carbon–carbon double bond of (2E)-enoyl-CoA substrates in the β-oxidation lipid-degrading pathway. The hydration reaction is very challenging to carry out by traditional chemical synthesis, as no selective catalysts are available. Structural enzymology of ECH and its complexes with (3S)-hydroxyacyl-CoA products show that hydrogen bonds between the adenine 6-amino group of the ADP moiety of CoA and loop-2 induce a small structural change in this active site loop, tightening the NN distance between the hydrogen bond donors of the oxyanion hole from 5.2 Å (unliganded) to 4.0 Å and forming a competent oxyanion hole at the catalytic site. A structurally similar and catalytically competent oxyanion hole is observed in the complex with (3S)-hydroxyhexanoyl PAN and the activator 3′,5′-ADP, both bound at the active site. The use of 3′,5′-ADP as the activator enables the synthetic use of ECH for the hydration of a wide range of (2E)-enoyl-PAN substrates with different steric demands and functionalities. The products, 3-hydroxyacyl-PAN thioesters, were obtained in good isolated yields and excellent stereoselectivities (typically >99:<1 3S:3R). Even for acyl chains that contain reactive groups such as bromide or methyl ester functionalities at C7, no side products resulting from potentially competing cyclization could be detected in the enzymatic hydration protocol.
Dalwani et al. (Mon,) studied this question.