Spectroscopic Investigation of Cysteamine Dioxygenase

Abstract

Thiol dioxygenases are mononuclear non-heme Fe II -dependent metalloenzymes that initiate the oxidative catabolism of thiol-containing substrates to their respective sulfinates. Cysteine dioxygenase (CDO), the best characterized mammalian thiol dioxygenase, contains a three-histidine (3-His) coordination environment rather than the 2-His-1-carboxylate facial triad seen in most mononuclear non-heme Fe II enzymes. A similar 3-His active site is found in the bacterial thiol dioxygenase 3-mercaptopropionate dioxygenase (MDO), which converts 3-mercaptopropionate into 3-sulfinopropionic acid as part of the bacterial sulfur metabolism pathway. In this study, we have investigated the active site geometric and electronic structures of a third non-heme Fe II -dependent thiol dioxygenase, cysteamine dioxygenase (ADO), by using a spectroscopic approach. Although a 3-His facial triad had previously been implicated on the basis of sequence alignment and site-directed mutagenesis studies, little is currently known about the active site environment of ADO. Our magnetic circular dichroism and electron paramagnetic resonance data provide compelling evidence that ADO features a 3-His facial triad, like CDO and MDO. Despite this similar coordination environment, spectroscopic results obtained for ADO incubated with various substrate analogues are distinct from those obtained for the other Fe II -dependent thiol dioxygenases. This finding suggests that the secondary coordination sphere of ADO is distinct from those of CDO and MDO, demonstrating the significant role that secondary-sphere residues play in dictating substrate specificity.

Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center

“…The arc consists of several underlying peaks with similar anisotropic hyperfine couplings. There are two additional sets of peaks with frequencies of ( 6 , 7 , 8 , 9 ) MHz; these are more weakly coupled 1 H as they appear closer to the 1 H Larmor frequency along the diagonal.…”

“…Thiol dioxygenases are a subset of nonheme mononuclear iron oxygenases that catalyze the O 2 -dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. Among this group, cysteine dioxygenase (CDO) ( 1 , 2 , 3 , 4 , 5 ) and cysteamine dioxygenase (ADO) ( 6 , 7 ) are responsible for the biosynthesis of cysteine sulfinic acid (CSA) and hypotaurine (HT), respectively, which are precursors for the biosynthesis of taurine ( 1 ). Among bacteria, a number of other thiol dioxygenases have been identified, including mercaptosuccinate dioxygenase (MSDO) ( 8 ) and 3-mercaptopropionate dioxygenase (3MDO) ( 9 , 10 , 11 , 12 , 13 , 14 ).…”

Emerging roles for thiol dioxygenases as oxygen sensors

“…The precise role of the alternative triad in NCOs is not yet clear, but an Asp176Asn variant in AtPCO4 resulted in near-ablated activity. If, as reported for ADO, substrate coordination of NCOs is monodentate to the active site metal [38], it is possible that the conserved Asp in the NCO form of this triad could play a role in binding the N-terminal amine of their polypeptide substrates.…”

“…It is also possible that ADO shares another structural feature with CDO: in 2018, Wang et al published evidence that a Cys-Tyr crosslink can occur in ADO between Cys 220 and Tyr 222 [37]. Interestingly, recent spectroscopic investigations indicate that metal -substrate coordination in ADO is distinctive from that of CDO: While crystallographic evidence has shown these enzymes to bind their substrates in a bidentate manner, spectroscopic evidence has indicated that ADO binds cysteamine in a monodentate manner via the thiol but not via its amine group [38,39].…”

Crystal structure of human cysteamine dioxygenase provides a structural rationale for its function as an oxygen sensor

“…Additionally, ADO can readily form dinitrosyl iron complexes anaerobically in the presence of substrates (21). EPR and magnetic circular dichroism characterization of the Fe(III)-ADO by Brunold et al has led to a similar conclusion of the substrate-binding mode and a distinct secondary coordination sphere from CDO and MDO (24).…”