Structural basis of a multi-functional deaminase in chlorovirus PBCV-1.

2-Deoxycytidylate deaminase (dCD) is a member of the zinc-dependent cytidine deaminase family features in its allosterically regulated mechanism by dCTP and dTTP. The large double-stranded DNA-containing chlorovirus PBCV-1 encodes a dCD family enzyme PBCV1dCD that was reported to be able to deaminize both dCMP and dCTP, which makes PBCV1dCD unique in the dCD family proteins. In this study, we report the crystal structure of PBCV1dCD in complex with dCTP/dCMP and dTTP/dTMP, respectively. We further proved the ability of PBCV1dCD in the deamination of dCDP, which makes PBCV1dCD a multi-functional deaminase. The structural basis for the versatility of PBCV1dCD is analyzed and discussed, with the finding of a unique Trp121 residue key to the deamination and substrate binding ability. Our findings may broaden the understanding of dCD family proteins and provide novel insights into the multi-functional enzyme.

Spectroscopic and calorimetric assays reveal dependence on dCTP and two metals (Zn2++Mg2+) for enzymatic activity of Schistosoma mansoni deoxycytidylate (dCMP) deaminase.

The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown deaminase function in this parasite.

Mechanism of the allosteric regulation of Streptococcus mutans 2'-deoxycytidylate deaminase.

In cells, dUMP is the intermediate precursor of dTTP in its synthesis during deoxynucleotide metabolism. In Gram-positive bacteria and eukaryotes, zinc-dependent deoxycytidylate deaminases (dCDs) catalyze the conversion of dCMP to dUMP. The activity of dCD is allosterically activated by dCTP and inhibited by dTTP. Here, the crystal structure of Streptococcus mutans dCD (SmdCD) complexed with dTTP is presented at 2.35 Šresolution, thereby solving the first pair of activator-bound and inhibitor-bound structures from the same species to provide a more definitive description of the allosteric mechanism. In contrast to the dTTP-bound dCD from the bacteriophage S-TIM5 (S-TIM5-dCD), dTTP-bound SmdCD adopts an inactive conformation similar to the apo form. A structural comparison suggests that the distinct orientations of the triphosphate group in S-TIM5-dCD and SmdCD are a result of the varying protein binding environment. In addition, calorimetric data establish that the modulators bound to dCD can be mutually competitively replaced. The results reveal the mechanism underlying its regulator-specific activity and might greatly enhance the understanding of the allosteric regulation of other dCDs.

Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides.

Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.

The first crystal structure of a dTTP-bound deoxycytidylate deaminase validates and details the allosteric-inhibitor binding site.

Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.

Crystal structures of Streptococcus mutans 2'-deoxycytidylate deaminase and its complex with substrate analog and allosteric regulator dCTP x Mg2+.

2'-Deoxycytidylate deaminase [or deoxycytidine-5'-monophosphate (dCMP) deaminase, dCD] catalyzes the deamination of dCMP to deoxyuridine-5'-monophosphate to provide the main nucleotide substrate for thymidylate synthase, which is important in DNA synthesis. The activity of this homohexameric enzyme is allosterically regulated by deoxycytidine-5'-triphosphate (dCTP) as an activator and by deoxythymidine-5'-triphosphate as an inhibitor. In this article, we report the crystal structures of dCMP deaminase from Streptococcus mutans and its complex with dCTP and an intermediate analog at resolutions of 3.0 and 1.66 A. The protein forms a hexamer composed of subunits adopting a three-layer alpha/beta/alpha sandwich fold. The positive allosteric regulator dCTP mainly binds at the interface between two monomers in a molar ratio of 1:1 and rearranges the neighboring interaction networks. Structural comparisons and sequence alignments revealed that dCMP deaminase from Streptococcus mutans belongs to the cytidine deaminase superfamily, wherein the proteins exhibit a similar catalytic mechanism. In addition to the two conserved motifs involved in the binding of Zn(2+), a new conserved motif, (G(43)YNG(46)), related to the binding of dCTP was also identified. N-terminal Arg4, a key residue located between two monomers, binds strongly to the gamma phosphate group of dCTP. The regulation signal was transmitted by Arg4 from the allosteric site to the active site via modifications in the interactions at the interface where the substrate-binding pocket was involved and the relocations of Arg26, His65, Tyr120, and Arg121 to envelope the active site in order to stabilize substrate binding in the complex. Based on the enzyme-regulator complex structure observed in this study, we propose an allosteric mechanism for dCD regulation.

Chloroviruses encode a bifunctional dCMP-dCTP deaminase that produces two key intermediates in dTTP formation.

The chlorovirus PBCV-1, like many large double-stranded DNA-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis. This report describes the characterization of the PBCV-1 dCMP deaminase, which produces dUMP, a key intermediate in the synthesis of dTTP. As predicted, the recombinant protein has dCMP deaminase activity that is activated by dCTP and inhibited by dTTP. Unexpectedly, however, the viral enzyme also has dCTP deaminase activity, producing dUTP. Typically, these two reactions are catalyzed by proteins in separate enzyme classes; to our knowledge, this is the first example of a protein having both deaminase activities. Kinetic experiments established that (i) the PBCV-1 enzyme has a higher affinity for dCTP than for dCMP, (ii) dCTP serves as a positive heterotropic effector for the dCMP deaminase activity and a positive homotropic effector for the dCTP deaminase activity, and (iii) the enzymatic efficiency of the dCMP deaminase activity is about four times higher than that of the dCTP deaminase activity. Inhibitor studies suggest that the same active site is involved in both dCMP and dCTP deaminations. The discovery that the PBCV-1 dCMP deaminase has two activities, together with a previous report that the virus also encodes a functional dUTP triphosphatase (Y. Zhang, H. Moriyama, K. Homma, and J. L. Van Etten, J. Virol. 79:9945-9953, 2005), means that PBCV-1 is the first virus to encode enzymes involved in all three known pathways to form dUMP.

Three-dimensional structure of the R115E mutant of T4-bacteriophage 2'-deoxycytidylate deaminase.

2'-Deoxycytidylate deaminase (dCD) converts deoxycytidine 5'-monophosphate (dCMP) to deoxyuridine 5'-monophosphate and is a major supplier of the substrate for thymidylate synthase, an important enzyme in DNA synthesis and a major target for cancer chemotherapy. Wild-type dCD is allosterically regulated by the end products of its metabolic pathway, deoxycytidine 5'-triphosphate and deoxythymidine 5'-triphosphate, which act as an activator and an inhibitor, respectively. The first crystal structure of a dCD, in the form of the R115E mutant of the T4-bacteriophage enzyme complexed with the active site inhibitor pyrimidin-2-one deoxyribotide, has been determined at 2.2 A resolution. This mutant of dCD is active, even in the absence of the allosteric regulators. The molecular topology of dCD is related to that of cytidine deaminase (CDA) but with modifications for formation of the binding site for the phosphate group of dCMP. The enzyme has a zinc ion-based mechanism that is similar to that of CDA. A second zinc ion that is present in bacteriophage dCD, but absent in mammalian dCD and CDA, is important for the structural integrity of the enzyme and for the binding of the phosphate group of the substrate or inhibitor. Although the R115E mutant of dCD is a dimer in solution, it crystallizes as a hexamer, mimicking the natural state of the wild-type enzyme. Residues 112 and 115, which are known to be important for the binding of the allosteric regulators, are found in a pocket that is at the intersubunit interfaces in the hexamer but distant from the substrate-binding site. The substrate-binding site is composed of residues from a single protein molecule and is sequestered in a deep groove. This groove is located at the outer surface of the hexamer but ends at the subunit interface that also includes residue 115. It is proposed that the absence of subunit interactions at this interface in the dimeric R115E mutant renders the substrate-binding site accessible. In contrast, for the wild-type enzyme, binding of dCTP induces an allosteric effect that affects the subunit interactions and results in an increase in the accessibility of the binding site.

Base-modification mRNA editing through deamination--the good, the bad and the unregulated.

RNA editing is a co- or post-transcriptional process in which select nucleotide sequences in RNA are altered from that originally encoded in the genome. The mRNAs encoding apolipoprotein B and some glutamate receptor subunits of ionotropic membrane channels are edited by site-specific base-deamination systems. Although these editing systems differ markedly in their mechanism for RNA-substrate binding and in their catalytic subunits, recent results suggest potentially common solutions to the problem of editing-site selectivity. The data suggest that there are multiple editing complexes or 'editosomes', which manifest editing-site preferences due to their macromolecular composition.

A novel zinc-binding motif found in two ubiquitous deaminase families.

Two families of deaminases, one specific for cytidine, the other for deoxycytidylate, are shown to possess a novel zinc-binding motif, here designated ZBS. We have (1) identified the protein members of these 2 families, (2) carried out sequence analyses that allow specification of this zinc-binding motif, and (3) determined signature sequences that will allow identification of additional members of these families as their sequences become available.

Identification of a site necessary for allosteric regulation in T4-phage deoxycytidylate deaminase.

An allosteric inhibitor of dCMP deaminase, dTTP, forms a photolabile covalent bond with T4-phage dCMP deaminase in the presence of UV light at 254 nm. The importance of the methyl group in this process is supported by the findings that dUTP, also an allosteric inhibitor, does not photofix to the enzyme and that tritium is released from [methyl-3H dTTP during the course of the photofixation. That the bond formed is photolabile is demonstrated by the fact that tritium is released by about 10-fold over the amount of nucleotide that is photofixed. The amino acid that covalently binds dTTP in T4-dCMP deaminase was identified as Phe112. On conversion of Phe112 to an alanine by site-directed mutagenesis, there was a dramatic change in the enzyme's response to its allosteric effectors when measured early in the reaction, in that the mutant enzyme was as active as the wild-type even in the absence of dCTP and was only weakly inhibited by dTTP. However, after 10-15% of the substrate had been deaminated, the reaction rate fell off rather markedly, indicating either that an inhibitor was being accumulated on the enzyme or that the enzyme was being irreversibly inactivated with time. That the latter was not the case was shown by the addition of dCTP to the reaction, which restored the rate to that expected when it was present initially. Furthermore, we showed that, consistent with the observed loss of allosteric regulation by dCTP and dTTP, the affinity of the mutant enzyme for dTTP and dCTP as determined by binding studies was greatly reduced relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

T4-phage deoxycytidylate deaminase is a metalloprotein containing two zinc atoms per subunit.

Deoxycytidylate (dCMP) deaminase, a hexameric allosteric enzyme induced on infection of Escherichia coli by bacteriophage T4, was shown to contain two atoms of zinc per subunit by atomic absorption spectroscopy. One zinc appears to be involved in catalysis, as described for adenosine deaminase (Sharaff, A. J., Wilson, D. K., Chang, Z., and Quiocho, F. A. (1992) J. Mol. Biol. 226, 917-921) and cytidine deaminase (Yang, C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). This thesis is supported by the finding that the enzyme loses about 80% of its activity in the presence of o-phenanthroline. It has also been found that zinc is released when the enzyme is denatured in the presence of the metallochromic indicator, 4-(2-pyridylazo)resorcinol. Renaturation of the deaminase to an active form occurred in the presence but not in the absence of zinc. The second atom of zinc is proposed to be located in a region of T4-dCMP deaminase that resembles a zinc finger. This region, which has the sequence His-X3-Cys-X14-His-X3-His, would represent a zinc-binding motif that has not been described previously.

Allosteric modifier and substrate binding of donkey deoxycytidylate aminohydrolase (EC 3.5.4.12)

The hexameric allosteric enzyme deoxycytidylate aminohydrolase from donkey spleen is shown by equilibrium dialysis to bind specifically the allosteric inhibitor, dTTP, the activator dCTP, and the substrate analog dAMP each at six sites (the dTTP and dCTP sites may or may not be identical). These conclusions contrast with earlier ones that there were four sites for each effector; reasons for the discrepancy are discussed. With the knowledge of site numbers and the kinetic information from the accompanying paper it is concluded that the kinetic cooperativity of the enzyme excludes a concerted conformational transition mechanism. Amino acid analysis gives a molecular weight of 18,842 Da per subunit, i.e., 113,052 for the hexamer. A new simplified purification of homogeneous enzyme from donkey spleen probably useful for dCMP aminohydrolase from other sources is described.