Proline dehydrogenase (PRODH) is
a flavoenzyme that catalyzes the
first step of proline catabolism, the oxidation of l-proline
to Δ1-pyrroline-5-carboxylate. PRODH has emerged
as a cancer therapy target because of its involvement in the metabolic
reprogramming of cancer cells. Here, we report the discovery of a
new class of PRODH inactivator, which covalently and irreversibly
modifies the FAD in a light-dependent manner. Two examples, 1,3-dithiolane-2-carboxylate
and tetrahydrothiophene-2-carboxylate, have been characterized using
X-ray crystallography (1.52–1.85 Å resolution), absorbance
spectroscopy, and enzyme kinetics. The structures reveal that in the
dark, these compounds function as classical reversible, proline analogue
inhibitors. However, exposure of enzyme-inhibitor cocrystals to bright
white light induces decarboxylation of the inhibitor and covalent
attachment of the residual S-heterocycle to the FAD N5 atom, locking
the cofactor into a reduced, inactive state. Spectroscopic measurements
of the inactivation process in solution confirm the requirement for
light and show that blue light is preferred. Enzyme activity assays
show that the rate of inactivation is enhanced by light and that the
inactivation is irreversible. We also demonstrate the photosensitivity
of cancer cells to one of these compounds. A possible mechanism is
proposed involving photoexcitation of the FAD, while the inhibitor
is noncovalently bound in the active site, followed by electron transfer,
decarboxylation, and radical combination steps. Our results could
lead to the development of photopharmacological drugs targeting PRODH.