Enantioselective Hydration of Non-CoA Enoyl-Thioesters by Enoyl-CoA Hydratase (ECH): Activation of the Active Site Oxyanion Hole with 3′,5′-Adenosine-Diphosphate Enables Competent Catalysis

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.