A Structure-Based Approach for Detection of Thiol Oxidoreductases and Their Catalytic Redox-Active Cysteine Residues

Abstract

Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases. Among the 20 amino acids commonly found in proteins, cysteine (Cys) is special in that it is present more often than other residues in functionally important locations within proteins. Some of these functions include metal binding, catalysis, structural stability, and posttranslational modifications. Identifying these functions in proteins of unknown function is difficult, in part because it is unclear which features distinguish one Cys function from the other. Among proteins with functionally important Cys, a large group of proteins utilizes this residue for redox catalysis. These proteins possess different folds and are collectively known as thiol oxidoreductases. In this work, we developed a procedure that allows recognition of these proteins by analyzing their structures or structural models. The method is based on the analyses of amino acid and secondary structure composition of Cys environment in proteins, their similarity to known thiol oxidoreductases, and calculations of Cys accessibility, reactivity, and location in predicted active sites. The procedure performed well on a set of test proteins and also selectively recognized thiol oxidoreductases by analyzing the Saccharomyces cerevisiae protein set. Thus, this study generated new information about the structural features of thiol oxidoreductases and may help to recognize these proteins in protein structure databases.