Structural Investigation of Substrate Binding and Oxygen Addition in Thiol Dioxygenases

Thiol dioxygenases (TDOs) are a family of unique non-heme iron-dependent metalloenzymes that catalyze the addition of both atoms of molecular oxygen to the thiol groups of their substrates.¹ Previously characterized members have shown several important roles in thiol and oxygen homeostasis among bacteria, mammals, and plants.² These enzymes belong to the cupin enzyme superfamily, which is characterized by a β-barrel fold and has two semi-conserved cupin binding motifs. TDOs adopt a 3-His coordination of the iron atom, which deviates from the more common 2-His-1-carboxylate binding motif seen in many non-heme ferrous oxygenases.^{3, 4} Of the handful of TDOs identified, cysteine dioxygenase is the most extensively studied and is the only TDO with a native substrate-bound structure. Other members of the TDO family include 3-mercaptopropionate dioxygenase, cysteamine dioxygenase, and plant cysteine oxidase. Although these enzymes exhibit the typical cupin structural features and share a common metal coordination sphere, our understanding regarding their substrate recognition and oxygen addition is lacking, prompting the need for more rigorous investigation of this enzyme family. During proteomic studies of the bacterium Variovorax paradoxus strain B4 that utilizes mercaptosuccinate as its sole carbon and sulfur source, another TDO was discovered, mercaptosuccinate dioxygenase (MSDO).^{5, 6}  Although MSDO has been confirmed to catalyze the conversion of mercaptosuccinate to sulfinosuccinate through the addition of molecular oxygen, the structure of MSDO is currently unknown, and only a preliminary investigation has been conducted.^{5, 7} Prediction of MSDO’s mechanism on substrate recognition has been precluded by the diverse structures of substrates that vary from one TDO to another. Understanding of how substrate coordinates to the active site of TDOs is limited as CDO is the only enzyme with a native substrate-bound structure. By further studying structurally and functionally lesser-known thiol dioxygenases such as MSDO, we can advance our understanding of this enzyme family, leading to such applications as drug treatment for thiol homeostasis-related disorders. Research regarding MSDO’s activity in V. paradoxus will aid in establishing methods of bioremediation and other biotechnological applications.

References 

(1) Stipanuk, M. H.; Simmons, C. R.; Andrew Karplus, P.; Dominy, J. E. Thiol dioxygenases: unique families of cupin proteins. Amino acids 2011, 41, 91-102.

(2) Perri, M.; Licausi, F. Thiol dioxygenases: from structures to functions. Trends in Biochemical Sciences 2024.

(3) Khuri, S.; Bakker, F. T.; Dunwell, J. M. Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteins. Molecular Biology and Evolution 2001, 18 (4), 593-605.

(4) Hegg, E. L.; Jr, L. Q. The 2‐His‐1‐carboxylate facial triad—an emerging structural motif in mononuclear non‐heme iron (II) enzymes. European Journal of Biochemistry 1997, 250 (3), 625-629.

(5) Brandt, U.; Schuermann, M.; Steinbuechel, A. Mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, from Variovorax paradoxus strain B4 is the key enzyme of mercaptosuccinate degradation. Journal of Biological Chemistry 2014, 289 (44), 30800-30809.

(6) Brandt, U.; Deters, A.; Steinbüchel, A. A jack-of-all-trades: 2-mercaptosuccinic acid. Applied microbiology and biotechnology 2015, 99, 4545-4557.

(7) Brandt, U.; Galant, G.; Meinert-Berning, C.; Steinbüchel, A. Functional analysis of active amino acid residues of the mercaptosuccinate dioxygenase of Variovorax paradoxus B4. Enzyme and microbial technology 2019, 120, 61-68.