Structure and mechanism of mouse cysteine dioxygenase (original) (raw)
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Crystal Structure of Mammalian Cysteine Dioxygenase
2006
Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteine sulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5-Å resolution, and these results confirm the canonical cupin -sandwich fold and the rare cysteinyltyrosine intramolecular cross-link (between Cys 93 and Tyr 157 ) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His 86 , His 88 , and His 140 ) and a water molecule. Attempts to acquire a structure with bound ligand using either cocrystallization or soaking crystals with cysteine revealed the formation of a mixed disulfide involving Cys 164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploration of the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.
Biochemistry, 2010
Cysteine dioxygenase (CDO) is a mononuclear non-heme Fe-dependent dioxygenase that catalyzes the initial step of oxidative cysteine catabolism. Its active site consists of an Fe(II) ion ligated by three histidine residues from the protein, an interesting variation on the more common 2-His-1-carboxylate motif found in many other non-heme Fe(II)-dependent enzymes. Multiple structural and kinetic studies of CDO have been carried out recently, resulting in a variety of proposed catalytic mechanisms; however, many open questions remain regarding the structure/ function relationships of this vital enzyme. In this study, resting and substrate-bound forms of CDO in the Fe(II) and Fe(III) states, both of which are proposed to have important roles in this enzyme's catalytic mechanism, were characterized by utilizing various spectroscopic methods. The nature of the substrate/active-site interactions was also explored using the cysteine analog selenocysteine (Sec). Our electronic absorption, magnetic circular dichroism, and resonance Raman data exhibit features characteristic of direct S (or Se) ligation to both the high-spin Fe(II) and Fe(III) active-site ions. The resulting Cys (or Sec)-bound species were modeled and further characterized using density functional theory computations to generate experimentally validated geometric and electronic structure descriptions. Collectively, our results yield a more complete description of several catalytically relevant species and provide support for a reaction mechanism similar to that established for many structurally related 2-His-1-carboxylate Fe(II)-dependent dioxygenases.
Gene, 2001
The murine gene encoding cysteine dioxygenase (CDO; EC 1.13.11.20), a key enzyme of l-cysteine metabolism, was isolated and characterized, and the proximal promoter was identified. A bacterial artificial chromosome mouse library was screened and a single clone containing the entire CDO gene was isolated. The murine CDO gene contains five exons and spans about 15 kb. The open reading frame is encoded within all five exons. All intron/exon splice junctions and all intron sizes are conserved with the rat CDO gene and are very similar to those of the human CDO gene. The primary transcriptional initiation site is located 213 bp upstream of the initiation ATG codon. The nucleotide sequence of the 5 0 -promoter region is highly conserved between the mouse and rat genes and contains a TATA-box-like sequence and GC boxes. A variety of consensus cis-acting elements were also identified in the 5 0 -flanking region. These included HNF-3b, HFH-1, HFH-2, HFH-3, C/EBP, and C/EBPb, all of which are consistent with the tissue-specific expression profiles of the gene. Gene reporter studies of the CDO 5 0 -region indicated the presence of an active promoter within the first 223 bp upstream of the transcriptional initiation site and the possible presence of repressor elements upstream of bp 2223. Northern blot analyses indicated that the CDO gene displays tissue-specific expression, with the highest mRNA level present in liver and with detectable levels found in kidney, lung, brain and small intestine. Western blot analyses indicated that CDO protein levels parallel mRNA levels. These results are consistent with the known function of CDO in whole-body cysteine homeostasis. q
Cysteine dioxygenase: a robust system for regulation of cellular cysteine levels
Amino Acids, 2009
Cysteine catabolism in mammals is dependent upon cysteine dioxygenase (CDO), an enzyme that adds molecular oxygen to the sulfur of cysteine, converting the thiol to a sulfinic acid known as cysteinesulfinic acid (3-sulfinoalanine). CDO is one of the most highly regulated metabolic enzymes responding to diet that is known. It undergoes up to 45-fold changes in concentration and up to 10fold changes in catalytic efficiency. This provides a remarkable responsiveness of the cell to changes in sulfur amino acid availability: the ability to decrease CDO activity and conserve cysteine when cysteine is scarce and to rapidly increase CDO activity and catabolize cysteine to prevent cytotoxicity when cysteine supply is abundant. CDO in both liver and adipose tissues responds to changes in dietary intakes of protein and/or sulfur amino acids over a range that encompasses the requirement level, suggesting that cysteine homeostasis is very important to the living organism.
Influence of cysteine 164 on active site structure in rat cysteine dioxygenase
JBIC Journal of Biological Inorganic Chemistry, 2016
Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme with unique structural features, namely an intramolecular thioether crosslink between cysteine 93 and tyrosine 157, and a disulfide between substrate L-cysteine and cysteine 164 in the entrance channel to the active site. We investigated how these posttranslational modifications affect catalysis through a kinetic, crystallographic and computational study. The enzyme kinetics of a C164S variant are identical to WT indicating that disulfide formation at C164 does not significantly impair access to the active site at physiological pH. However, at high pH the cysteine-tyrosine crosslink formation is enhanced in C164S. This supports the view that disulfide formation at position 164 can limit access to the active site. The C164S yielded crystal structures of unusual clarity in both resting state and with cysteine bound. Both show that the iron in the cysteine bound complex is a mixture of penta-and hexa-coordinate with a water molecule taking up the final site (60% occupancy), which is where dioxygen is believed to coordinate during turnover. The serine also displays stronger hydrogen bond interactions to a water bound to the amine of the substrate cysteine. However, the interactions between cysteine and iron appear unchanged. DFT calculations support this and show that WT and C164S have similar binding energies for the water molecule in the final site. This variant therefore provides evidence that WT also exists in this equilibrium between penta-and hexa-coordinate forms and presence of the sixth ligand does not strongly affect dioxygen binding.
Probes of the Catalytic Site of Cysteine Dioxygenase
Journal of Biological Chemistry, 2006
The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the ␣-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ ␣-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by ␣-ketoglutarate.
Thiol dioxygenases: unique families of cupin proteins
Amino Acids, 2011
Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a 6-stranded β-barrel core, and this canonical cupin or "jelly roll" β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative fingerprint motif for ADOs, or DUF1637 family members, is proposed. In ADOs, the conserved glutamate residue in cupin motif 1 is replaced by either glycine or valine. Both ADOs and CDOs appear to represent unique clades within the cupin superfamily.
Journal of Molecular Biology, 2016
In mammals, the non-heme iron enzyme cysteine dioxygenase (CDO) helps regulate cysteine (Cys) levels through converting Cys to cysteine sulfinic acid (CSA). Its activity is in part modulated by the formation of a Cys93-Tyr157 crosslink that increases its catalytic efficiency over 10-fold. Here, 21 high-resolution mammalian CDO structures are used to gain insight into how the Cys-Tyr crosslink promotes activity and how select competitive inhibitors bind. Crystal structures of crosslink-deficient C93A and Y157F variants reveal similar ~1.0 Å shifts in the side chain of residue 157, and both variant structures have a new chloride ion coordinating the active site iron. Cys binding is also different than for wild-type CDO and no Cys-persulfenate forms in the C93A or Y157F active sites at either pH=6.2 or 8.0. We conclude that the crosslink enhances activity by positioning the Tyr157 hydroxyl for enabling proper Cys binding, proper oxygen binding, and optimal chemistry. In addition, structures are presented for homocysteine, thiosulfate and azide bound as competitive inhibitors. The observed binding modes of homocysteine and D-Cys make clear why they are not substrates, and the binding of azide shows that, in contrast to what has been proposed, it does not bind in these crystals as a superoxide mimic.