Evaluation of Enzymes in the Sulfur Metabolic Pathway of Mammals and Bacteria

Abstract

Sulfur-containing molecules are essential for all organisms. Mammals depend on the availability of amino acid methionine and cysteine, whereas bacteria rely on sulfite and cysteine. The research presented in this dissertation focuses on enzymes found within the sulfur metabolic pathway. These enzymes play an important role in cysteine oxidation in mammals and sulfite acquisition in bacteria. Cysteine dioxygenase (CDO) is a mononuclear non-heme iron enzyme that belongs to the cupin superfamily. CDO catalyzes the conversion of L-cysteine to cysteine sulfinic acid in the presence of dioxygen. Several cupin enzymes contain two partially conserved motifs, GX5HXHX3-6EX6G (motif 1) and GX5-7PXGX2HX3N (motif 2), that represent the 3-His/1-Glu coordination of the metal center. In mammalian CDO the traditional glutamate residue from the first motif is replaced with a cysteine (Cys93). Substitution of Cys93 with glutamate was shown to inhibit dioxygen consumption. The three dimensional structure of C93E CDO showed glutamate coordinates the iron similar to traditional 3-His/1-Glu cupins. Specifically, glutamate occupies the ligand site that is reserved for dioxygen, and therefore cysteine oxidation cannot occur. Incorporation of the cysteine residue frees the ligand site promoting bidentate coordination of the L-cysteine substrate and single site dioxygen binding. Furthermore, replacement of glutamate with cysteine allows for the formation of a unique thioether crosslink with a nearby tyrosine residue (Tyr157). In wild-type CDO the crosslink exists as a heterogeneous mixture of non crosslinked and crosslinked isoforms. In CDO crosslink formation is dependent on iron, dioxygen, and L-cysteine. However the Cys-Tyr crosslink in other enzymes is not dependent on the substrate. It is unusual that crosslink formation in CDO is dependent on the L-cysteine substrate. To understand the role of L-cysteine in crosslink formation, wild-type CDO was incubated with L-cysteine analogs. In addition to L-cysteine, wild-type CDO is able to form the crosslink with D-cysteine, but is unable to form the crosslink with cysteamine and 3-mercaptopropionic acid. However, previous studies found the CDO variant R60A was able to form the crosslink with cysteamine. Through electrostatic interactions Arg60 plays an important role in substrate stabilization. Evaluation of non crosslinked R60A CDO suggests that Arg60 also plays a role in substrate specificity for crosslink formation. In addition, non crosslinked R60A was able to form the crosslink with D-cysteine. These findings suggests that crosslink formation in cysteine dioxygenase is not specific to L-cysteine, but can occur with any small molecule capable of promoting dioxygen binding. Currently all proposed mechanisms for crosslink formation are based on the formation of a hydroxyl radical from Tyr157. Three dimensional analysis of non crosslinked wild-type CDO suggests that crosslink formation could also occur via a thiol radical from Cys93. Bacterial organisms require sulfur for survival, but sulfur is often limiting in the environment. However, bacteria possess several sets of enzymes capable of scavenging sulfite from organosulfate molecules. Two proteins expressed during sulfur limiting conditions in E. coli are a FMN-reductase SsuE and an alkanesulfonate monooxygenase SsuD. SsuD utilizes reduced flavin, supplied by SsuE, to cleave the carbon-sulfur bond of 1-alkanesulfonates into sulfite and the corresponding aldehyde. Flavin transfer between SsuE and SsuD occurs through a channeling mechanism involving protein-protein interactions. The alkanesulfonate monooxygenase system is one of several identified FMN-dependent two-component systems activated during sulfur starvation. In addition to Ssu, Msu and Sfn systems have been identified in Pseudomonas sp.. Together these enzymes catabolize dimethyl sulfone into inorganic sulfite. A high amino acid sequence identity exists between the reductases and monooxygenases involved in sulfur acquisition, and may utilize similar mechanistic approaches for flavin transfer. Initial desulfonation assays with SsuE and SsuD from P. putida do not support flavin transfer by a channeling mechanism. However, further investigation into the mechanism of flavin transfer for Msu and Sfn enzymes could provide evidence to support a channeling mechanism.