This Is AuburnElectronic Theses and Dissertations

Investigating the Catalytic and the Metabolic Role of the Thiol Dioxygenase and a Sulfurtransferase in the mdo Operon from Pseudomonas aeruginosa

Date

2019-12-10

Author

Laura, Tombrello

Type of Degree

PhD Dissertation

Department

Chemistry and Biochemistry

Restriction Status

EMBARGOED

Restriction Type

Auburn University Users

Date Available

12-01-2022

Abstract

Mammalian cysteine dioxygenase (CDO) is a mononuclear iron containing enzyme that belongs to the cupin superfamily. CDO catalyzes the oxidation of cysteine to cysteine sulfinic acid. It contains an amino acid-derived cofactor formed between residues Cys93-Tyr157. Reported CDO homologs have been identified in several bacteria; however, all known bacterial thiol dioxygenase enzymes lack the Cys-Tyr post-translational modification because of a highly conserved Gly in place of Cys93.1 Bacterial thiol dioxygenase enzymes are subdivided into the Arg-type and Gln-type, based on the identity of a conserved active site residue involved in substrate binding. The Arg-type has an alleged substrate specificity for L-cysteine and the Gln-type has a putative specificity for 3-mercaptopropionate (3-MPA).2 The Gln-type CDO enzyme homologs convert 3-MPA to 3-sulfinopropionate (3-SPA). However, the metabolic role of either 3-MPA or 3-SPA is unclear in these bacteria systems. The Gln-type enzymes are currently referred to as 3-mercaptopropionate dioxygenase (MDO). Substitutions of Cys93, Gln62, and Arg60 were made to evaluate the functional role and substrate specificity among thiol dioxygenases. The C93G CDO variant was unable to form the crosslink, and only showed a slight reduction in the catalytic efficiency with L-cysteine. The results from the studies performed with R60Q and Q62R, suggest that Arg60 plays a more defined role in substrate specificity in mammalian CDO than the comparable glutamine residue does in bacterial MDO. In bacteria that express “Gln-type” MDO, the gene is on the same operon as an annotated sulfurtransferase, but the existence of the sulfurtransferase has not been recognized by groups working with MDO. As both genes are located on the same operon, they likely catalyze reactions in a common metabolic pathway. Both the MDO and sulfurtransferase genes are located in low-sulfur islands, suggesting these genes are turned on when sulfur is limiting. The annotated sulfurtransferase on the mdo operon has an amino acid sequence similarity to mercaptopyruvate sulfurtransferase enzymes, which suggest that mercaptopyruvate could be a potential substrate for MDO or the sulfurtransferase. Kinetic studies were repeated for R60Q CDO and Q62R MDO using 3-mercaptopyruvate as a substrate. The increase of activity with wild-type MDO suggest that 3-mercaptopyruvate is a viable substrate for bacterial thiol dioxygenase, which is further justified by the decrease in catalytic activity for Q62R MDO. Natural fusions between thiol dioxygenases and sulfurtransferases have also been identified in certain bacteria. Existence of these fusions suggest that interactions among the dioxygenase and the sulfurtransferase could occur. Therefore, protein-protein interaction studies were performed with MDO and the sulfurtransferase. Our studies suggest that protein-protein interactions are occurring between MDO and the sulfurtransferase. The mdo operon in Pseudomonas aeruginosa is located in the same gene cluster as sulfur starvation enzymes. In order to determine if the mdo operon was regulated under sulfur limitation, growth and expression assays were performed on transposon insertions of each gene and analyzed with various sulfur sources. However, based on the results of the studies, the mdo operon is not involved in sulfur starvation. Instead, the mdo operon may be involved in hydrogen sulfide or cyanide detoxification. The studies described herein evaluated the functional role and substrate specificity of MDO and the sulfurtransferase in P. aeruginosa. Gln62 appears to play a role in substrate specificity but it does not seem to be as important as the role of Arg60 in mammalian CDO. Based on the evidence provided, MDO and the sulfurtransferase are able to interact. However, based on the results the mdo operon is more likely to be involved in hydrogen sulfide or cyanide detoxification rather than sulfur limitation. These studies also provided a foundation for future studies regarding the mdo operon in P. aeruginosa.