Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of Activity

Abstract

Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the ␥-sulfur of residue cysteine 93 and the aromatic side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examined what effect cofactor formation had on activity in vitro. Like other cross-linked cofactorcontaining enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe 2؉ ) and O 2 . Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiologically relevant cysteine concentrations, cofactor formation increased CDO catalytic efficiency by ϳ10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiological consequences.Recent biochemical and crystallographic studies have revealed that some enzymes contain unusual modifications to the amino acid residues residing within their active sites. Also known as amino acid-derived cofactors, these altered residues represent a new and exciting area for research in the field of protein post-translational modifications. Unlike other posttranslational modifications, which are made by attaching extrinsic molecules onto target proteins through reactions that are specifically catalyzed by third party enzymes, amino acid cofactors are generated directly from the amino acids within the proteins that contain them and are required for the production of fully competent enzymes (1, 2). From a biological perspective, amino acid-derived cofactors are significant because they can create novel structural motifs within the enzyme active site as well as alter the chemical properties of the unmodified parent amino acid residues and thus expand the otherwise limited range of catalytic reactions in which the common amino acids can participate.One recent addition to the list of enzymes known to contain an amino acid cofactor is cysteine dioxygenase (CDO).3 CDO is an iron (Fe 2ϩ )-dependent thiol dioxygenase that uses molecular oxygen to oxidize the sulfhydryl group of cys...

Influence of cysteine 164 on active site structure in rat cysteine dioxygenase

“…This reduction in activity has been shown in both human [16] and rat CDO, [18] but the size of the decrease reported is quite different, depending on substrate concentration. To resolve seemingly conflicting statements about the role of cysteine 164, we re-investigated the C164S variant to explore what changes in activity could be observed and to understand if these could be explained structurally.…”

“…Once formed, the disulfide at position 164 impedes access to the active site and thus abrogates crosslink formation, which has been shown to require cysteine. [15,18] Conversely, it could be the negative charge on the thiolate itself that might impede access. Either way, the C164S variant is unable to deprotonate in this pH range and/or form this disulfide and therefore is able to be fully crosslinked, in contrast to WT.…”

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase

“…CDO belongs to a family of non-heme mononuclear iron dioxygenases but stands out due to a number of unique characteristics. The iron atom has His 3 coordination, and, in mammalian forms, a post-translational modification involving a cysteine-to-tyrosine cross-link is present (24,25).…”

Expression of genes, encoding the enzymes of cysteine metabolism in human placenta in the first and third trimesters of uncomplicated pregnancy

“…It is known that CDO is highly expressed in the liver, adipocytes and term placenta [http://www.genecards.org/cgi-bin/ carddisp.pl?gene=CDO1&keywords=cysteine, dioxygenase#expression]. CDO is the most intensively regulated metabolic enzyme [28,29]. In response to the increased consumption of protein and sulfur-containing amino acids its concentration increases for several hours about 30 to 45 times [28] and its catalytic activity nearly 10 times [29].…”

“…CDO is the most intensively regulated metabolic enzyme [28,29]. In response to the increased consumption of protein and sulfur-containing amino acids its concentration increases for several hours about 30 to 45 times [28] and its catalytic activity nearly 10 times [29]. Thus, altogether CDO activity increases 300 to 450 times.…”