Facilitated By

San Antonio Medical Foundation

Probing Cofactor Biogenesis and Catalytic Mechanism in Iron and Copper Proteins Using Genetically-Substituted Noncanonical Amino Acids

The University of Texas at San Antonio

The University of Texas at San Antonio is an emerging Tier One research institution with nearly 29,000 students.

Principal Investigator(s)
Aimin Liu
Funded by
Natl Science Fdn
Research Start Date

The protein-derived cysteine-tyrosine (Cys-Tyr) redox cofactor is present in a growing number of iron- and copper-containing proteins. Its presence enables or significantly enhances the capacity of the host metalloprotein to mediate a specific redox process. However. the mechanism of Cys-Tyr biosynthesis remains poorly understood due to several intrinsic challenges. particularly its formation from self-processing due to uncoupled oxidation reactions in non-heme iron-dependent enzymes. This proposal is concerned with the mechanism of Cys-Tyr biogenesis in two iron-dependent human thiol dioxygenases. A copper-dependent oxidase from a bacterial source is included in the study for comparison. The human thiol dioxygenases. cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO). play a central role in the biological portion of the sulfur cycle and thiol metabolism. The catalytic activity of these enzymes is important in controlling cysteine and taurine levels. CDO contains a self-processed Cys-Tyr cofactor. It has been a long-standing question whether or not ADO also contains a Cys-Tyr cofactor. One common feature known so far in Cys-Tyr biogenesis in these proteins is that the formation of the thioether (C-S) bond is a metal-dependent process. The work proposed here will utilize site-specific genetic incorporation of unnatural amino acids. protein X-ray crystallography. analytic and spectroscopic studies to (1) determine the molecular basis of how the iron-activated oxidant is directed for cofactor biogenesis in CDO and the role of the Cys-Tyr cofactor in thiol dioxygenation. (2) determine the structure and enzymatic mechanism of ADO. and (3) investigate how the Cys-Tyr radical cofactor is formed in copper-dependent galactose oxidases (GAO) using genetic incorporation of unnatural amino acids to the tyrosine. These studies will elucidate the unknown mechanism of metallocenter-mediated sulfur oxidation and cofactor biogenesis. A better understanding of the irreversible self-processing C-S bond protein modification will also help to meet the fundamental challenge of predicting protein structures and functions.

Collaborative Project
Basic Research