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...

There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNAmediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity.There are many different processes in mammalian cells that result in the oxidation of thiol groups. Because of their reactivity, free sulfhydryl groups are highly susceptible to oxidation that results in the formation of disulfides, sulfenates, sulfinates, and sulfonates. Many of these reactions occur nonenzymatically, principally as a consequence of adventitious free radicals arising from aerobic respiration. Nevertheless, there are a small number of thiol oxidation reactions that are known to occur directly via enzymatic catalysis. The enzymes that catalyze these reactions show a high degree of substrate specificity and confer to cells the advantage of being able to precisely regulate the level of a particular reduced thiol.One interesting subset of the enzymes capable of specifically oxidizing free sulfhydryl groups are the thiol dioxygenases. In mammals this family comprises only two known proteins: cysteine dioxygenase (CDO, 3 EC 1.13.11.20) and cysteamine dioxygenase (EC 1.13.11.19). CDO adds two atoms of oxygen to free cysteine to yield cysteine sulfinic acid, whereas cysteamine dioxygenase adds two atoms of oxygen to free cysteamine (2-aminoethanethiol) to form hypotaurine (Fig. 1). The activities for these two proteins were first reported in mammalian tissues almost 40 years ago (1, 2). Since that time, howev...

The strong association of Zika virus infection with congenital defects has led to questions of how a flavivirus is capable of crossing the placental barrier to reach the fetal brain. Here, we demonstrate permissive Zika virus infection of primary human placental macrophages, commonly referred to as Hofbauer cells, and placental villous fibroblasts. We also demonstrate Zika virus infection of Hofbauer cells within the context of the tissue ex vivo using term placental villous explants. In addition to amplifying infectious virus within a usually inaccessible area, the putative migratory activities of Hofbauer cells may aid in dissemination of Zika virus to the fetal brain. Understanding the susceptibility of placenta-specific cell types will aid future work around and understanding of Zika virus–associated pregnancy complications.

Summary Tyro3, Axl and Mertk (TAM) receptors are candidate entry receptor for infection of Zika virus (ZIKV), an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we employed several routes of viral inoculation and compared viral replication in wild-type vs. Axl−/−, Mertk−/−, Axl−/−Mertk−/−, and Axl−/−Tyro3−/− mice in various organs. Pregnant and non-pregnant mice treated with interferon α receptor (IFNAR)-blocking (MAR1-5A3) antibody infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism was observed. These findings indicate that in mice, TAM receptors are not required for ZIKV entry and infection.

Zika virus replicates in mouse testes and causes testicular atrophy, with implication on sexual transmission and male fertility.