The role of outer-sphere residues and substrate-binding at the 3-mercaptopropionic acid dioxygenase (3mdo) active site: a combined spectroscopic and computational investigation

Thiol Dioxygenases (TDOs) are non-heme iron enzymes that catalyze the O2-dependent oxidation of thiol-bearing amino acid derivatives to their corresponding sulfinic acid. Recently, considerable work has been focused on this class of enzymes, as sulfur metabolite imbalances have been correlated with neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and motor neuron disease. The active site of this enzyme class is comprised of a mononuclear Fe(II) bound by three protein derived histidine residues. A conserved feature among structurally characterized TDOs is a sequence of serine, histidine, and tyrosine residues adjacent to the iron active site, deemed the SHY motif. By far, the most well studied TDO is mammalian cysteine dioxygenase (CDO) which oxidizes L-cysteine to cysteine sulfinic acid with high substrate-specificity. Unlike CDO, bacterial 3-mercaptopropionate dioxygenase from Azotobacter vinelandii (Av3MDO) is a promiscuous TDO that oxidizes a variety of thiol substrates. Given both enzymes show similar active site geometries, the drastic difference in substrate specificity is not well understood. This dissertation aims to address two unresolved topics among Av3MDO and TDOs in general. The first is the mode in which native substrate, 3MPA, binds to Av3MDO. Arguments have been made for both bidentate coordination through the substrate thiolate and carboxylate and monodentate through the thiolate only. Herein, crystallographic, spectroscopic, and computational studies are used to determine the mode of 3MPA coordination to the iron active site. Bidentate coordination was observed for a crystal structure of bound inhibitor, 3- hydroxypropionic acid, which was used as a basis for computational models of bidentate 3MPA coordination. These models showed agreement with spectroscopic data using either nitric oxide or cyanide as spectroscopic probes. The second aspect investigated is the role of the SHY motif in the active site. For Av3MDO, alterations to the SHY motif have been shown to drastically attenuate activity (kcat), catalytic efficiency (kcat/KM), and formation of NO-bound ES complex. However, the interactions between the SHY motif and iron active site are not fully understood. Spectroscopic studies presented herein reveal the flexible hydrogen bonding capabilities of Tyr159 in the SHY motif and its direct influence on the electronic structure of the iron active site.

Grantor dc:publisher

University of Alabama Libraries

Year dc:date.issued

2021

Author dc:creator

• York, Nicholas James

Advisor dc:contributor.advisor

• Pierce, Brad S.

Contributors dc:contributor

• Vincent, John B.
• Papish, Elizabeth T.
• Frantom, Patrick A.
• Summers, Ryan M.

• Chemistry
• Biochemistry
• Inorganic chemistry

Statement dc:rights

• All rights reserved by the author unless otherwise indicated.

Language dc:language.iso

en_US, English

Dc Identifier Other

u0015_0000001_0003849
York_alatus_0004D_14472

OAI identifier oai:identifier

oai:ir.ua.edu:123456789/7928