Cytosine residues in mammalian DNA occur in five forms, cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions1–4. The Tet family of dioxygenases is widely distributed across the tree of life5, including the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria6 encodes homologs of mammalian DNA methyltransferase and Tet proteins7. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 complexed with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active site pocket with planar stacking contacts, Watson–Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

Modified DNA bases in mammalian genomes, such as 5-methylcytosine ( 5m C) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic conversion of 5m C to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like 5m C oxygenase (NgTET1) from Naegleria gruberi, a single-celled protist evolutionarily distant from vertebrates. Here we show that NgTET1 is a 5-methylpyrimidine oxygenase, with activity on both 5m C (major activity) and thymidine (T) (minor activity) in all DNA forms tested, and provide unprecedented evidence for the formation of 5-formyluridine ( 5f U) and 5-carboxyuridine ( 5ca U) in vitro. Mutagenesis studies reveal a delicate balance between choice of 5m C or T as the preferred substrate. Furthermore, our results suggest substrate preference by NgTET1 to 5m CpG and TpG dinucleotide sites in DNA. Intriguingly, NgTET1 displays higher T-oxidation activity in vitro than mammalian TET1, supporting a closer evolutionary relationship between NgTET1 and the base J-binding proteins from trypanosomes. Finally, we demonstrate that NgTET1 can be readily used as a tool in 5m C sequencing technologies such as single molecule, realtime sequencing to map 5m C in bacterial genomes at base resolution.odified DNA bases exist in all forms of life, from viruses to mammals with many different biological roles. Accordingly, diverse mechanisms have evolved to "write," "read," and "erase" these modifications. In mammals, 5-methylcytosine ( 5m C) is the major form of DNA modification and is implicated in many crucial developmental processes. In human and mouse, 5m C can be successively oxidized into 5-hydroxymethylcytosine ( 5hm C), 5-formylcytosine ( 5f C), and 5-carboxylcytosine ( 5ca C) by the teneleven translocation (TET) family of oxygenases (1-4). The bases of 5f C and 5ca C can be excised by thymine DNA glycosylase (4). The 5m C-oxidation-coupled base-excision repair pathway provides a plausible route for active demethylation in mammalian cells. Many other species, from simple to complex, maintain DNA methylation machinery throughout their life cycle that may contribute to epigenetic regulation. Therefore, an interesting perspective is to examine shared and distinct features of TET oxygenases in diverse eukaryotes (5, 6).The human and mouse genomes encode three paralogous TET proteins, TET1, TET2, and TET3, which presumably carry out both redundant and distinct functions (7,8). TET proteins belong to the diverse group of α-ketoglutarate (αKG) and Fe(II)-dependent oxygenases (5). Subgroup classification based on sequence similarity links the TET proteins to base J-binding proteins (JBP1 and JBP2), which are primarily present in trypanosomes and possess thymidine (T)-hydroxylation activity (1). Further bioinformatic analysis revealed eight paralogous TET/ JBP-like genes in the genome of Naegleria gruberi, a single-celled amoeboflagellate protist that is a distant cousin of the par...

Protein farnesytransferase (FTase) catalyzes the transfer of a 15-carbon prenyl group from farnesyl diphosphate (FPP) to the cysteine residue of target proteins and is a member of the newest class of zinc metalloenzymes that catalyze sulfur alkylation. Common substrates of FTase include oncogenic Ras proteins, and therefore inhibitors are under development for the treatment of various cancers. An increased understanding of the salient features of the chemical transition state of FTase may aid in the design of potent inhibitors and enhance our understanding of the mechanism of this class of zinc enzymes. To investigate the transition state of FTase we have used transient kinetics to measure the alpha-secondary 3H kinetic isotope effect at the sensitive C1 position of FPP. The isotope effect for the FTase single turnover reaction using a peptide substrate that is farnesylated rapidly is near unity, indicating that a conformational change, rather than farnesylation, is the rate-limiting step. To look at the chemical step, the kinetic isotope effect was measured as 1.154 +/- 0.006 for a peptide that is farnesylated slowly, and these data suggest that FTase proceeds via a concerted mechanism with dissociative character.

The cell has >60 different farnesylated proteins. Many critically important signal transduction proteins are post-translationally modified with attachment of a farnesyl isoprenoid catalyzed by protein farnesyltransferase (FTase). Recently, it has been shown that farnesyl diphosphate (FPP) analogues can alter the peptide substrate specificity of FTase. We have used combinatorial screening of FPP analogues and peptide substrates to identify patterns in FTase substrate selectivity. Each FPP analogue displays a unique pattern of substrate reactivity with the tested peptides; FTase efficiently catalyzes the transfer of an FPP analogue selectively to one peptide and not another. Furthermore, we have demonstrated that these analogues can enter cells and be incorporated into proteins. These FPP analogues could serve as selective tools to examine the role prenylation plays in individual protein function.Mutant Ras proteins are one of the most important classes of oncogene products and are thus logical targets for cancer chemotherapeutics. Ras, both mutant and normal forms, must be farnesylated by protein farnesyltransferase (FTase) for proper processing, subcellular localization, and thus biological activity (Figure 1). Therefore, significant effort has been focused on the development of small-molecule FTase inhibitors (FTIs) as anticancer, antiRas therapeutics. Two FTIs are in advanced clinical trials (1). The clinical data have demonstrated, however, that FTIs do not function as anti-Ras agents, because K-Ras is alternatively prenylated by geranylgeranyltransferase I upon FTI treatment (23). The cellular (4) and clinical (1) efficacy of FTIs does not correlate with Ras mutational status. The FTI effectiveness observed in non-Ras-positive tumor cells is presumably elicited via inhibition of the farnesylation of other proteins crucial to the growth of tumors. This has led to significant interest in defining the entire set of mammalian prenylated proteins and determining their biological roles (5).Many proteins bearing a Ca 1 a 2 X sequence at their carboxyl terminus are modified by FTase using the C 15 isoprenoid farnesyl diphosphate (FPP) as a co-substrate. Substrate prediction models have estimated that there are >60 farnesylated cellular proteins (67), containing a wide variety of C-terminal sequences, and the inhibition of farnesylation of any individual or a combination of these proteins could be responsible for the antitumor effects of FTI treatment. The investigation of potential "protein-X" FTI targets has uncovered several proteins whose inactivation upon FTI treatment led to profound cellular consequences (8). Correspondingly, the investigation of the role of the farnesyl group on cellular proteins has been aided by the development of FTIs. However, using FTIs to investigate the function of the farnesyl lipid for an individual protein is cumbersome, as they are nonspecific tools. FTIs presumably block the farnesylation of all FTase substrate proteins in mammalian cells. Chemical agents that are capable ...

The kinetic mechanism of activation of the mitochondrial NAD-malic enzyme from the parasitic roundworm Ascaris suum has been studied using a steady-state kinetic approach. The following conclusions are suggested. First, malate and fumarate increase the activity of the enzyme in both reaction directions as a result of binding to separate allosteric sites, i.e., sites that exist in addition to the active site. The binding of malate and fumarate is synergistic with the K(act) decreasing by >or=10-fold at saturating concentrations of the other activator. Second, the presence of the activators decreases the K(m) for pyruvate 3-4-fold, and the K(i) (Mn) >or=20-fold in the direction of reductive carboxylation; similar effects are obtained with fumarate in the direction of oxidative decarboxylation. The greatest effect of the activators is thus expressed at low reactant concentrations, i.e., physiologic concentrations of reactant, where activation of >or=15-fold is observed. A recent crystallographic structure of the human mitochondrial NAD malic enzyme [13] shows fumarate bound to an allosteric site. Site-directed mutagenesis was used to change R105, homologous to R91 in the fumarate activator site of the human enzyme, to alanine. The R105A mutant enzyme exhibits the same maximum rate and V/K(NAD) as does the wild-type enzyme, but 7-8-fold decrease in both V/K(malate) and V/K(Mg), indicating the importance of this residue in the activator site. In addition, neither fumarate nor malate activates the enzyme in either reaction direction. Finally, a change in K143 (a residue in a positive pocket adjacent to that which contains R105), to alanine results in an increase in the K(act) for malate by about an order of magnitude such that it is now of the same magnitude as the K(m) for malate. The K143A mutant enzyme also exhibits an increase in the K(act) for fumarate (in the absence of malate) from 200 microM to about 25 mM.