The Methanococcus jannaschiidCTP Deaminase Is a Bifunctional Deaminase and Diphosphatase

Most bacteria produce the dUMP precursor for thymine nucleotide biosynthesis using two enzymes: a dCTP deaminase catalyzes the formation of dUTP and a dUTP diphosphatase catalyzes pyrophosphate release. Although these two hydrolytic enzymes appear to catalyze very different reactions, they are encoded by homologous genes. The hyperthermophilic archaeon Methanococcus jannaschii has two members of this gene family. One gene, at locus MJ1102, encodes a dUTP diphosphatase, which can scavenge deoxyuridine nucleotides that inhibit archaeal DNA polymerases. The second gene, at locus MJ0430, encodes a novel dCTP deaminase that releases dUMP, ammonia, and pyrophosphate. Therefore this enzyme can singly catalyze both steps in dUMP biosynthesis, precluding the formation of free, mutagenic dUTP. Besides differing from the previously characterized Salmonella typhimurium dCTP deaminase in its reaction products, this archaeal enzyme has a higher affinity for dCTP and its steady-state turnover is faster than the bacterial enzyme. Kinetic studies suggest: 1) the archaeal enzyme specifically recognizes dCTP; 2) dCTP deamination and dUTP diphosphatase activities occur independently at the same active site, and 3) both activities depend on Mg2+. The bifunctional activity of this M. jannaschii enzyme illustrates the evolution of a suprafamily of related enzymes that catalyze mechanistically distinct reactions. Most bacteria produce the dUMP precursor for thymine nucleotide biosynthesis using two enzymes: a dCTP deaminase catalyzes the formation of dUTP and a dUTP diphosphatase catalyzes pyrophosphate release. Although these two hydrolytic enzymes appear to catalyze very different reactions, they are encoded by homologous genes. The hyperthermophilic archaeon Methanococcus jannaschii has two members of this gene family. One gene, at locus MJ1102, encodes a dUTP diphosphatase, which can scavenge deoxyuridine nucleotides that inhibit archaeal DNA polymerases. The second gene, at locus MJ0430, encodes a novel dCTP deaminase that releases dUMP, ammonia, and pyrophosphate. Therefore this enzyme can singly catalyze both steps in dUMP biosynthesis, precluding the formation of free, mutagenic dUTP. Besides differing from the previously characterized Salmonella typhimurium dCTP deaminase in its reaction products, this archaeal enzyme has a higher affinity for dCTP and its steady-state turnover is faster than the bacterial enzyme. Kinetic studies suggest: 1) the archaeal enzyme specifically recognizes dCTP; 2) dCTP deamination and dUTP diphosphatase activities occur independently at the same active site, and 3) both activities depend on Mg2+. The bifunctional activity of this M. jannaschii enzyme illustrates the evolution of a suprafamily of related enzymes that catalyze mechanistically distinct reactions. dCTP deaminase dUTP diphosphatase pyrophosphate N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid 2-(N-cyclohexylamino)ethanesulfonic acid 3-(N-morpholino)propanesulfonic acid matrix-assisted laser desorption/ionization-time of flight trimethylsilyl electron impact-mass spectrometry Deoxyuridine nucleotides pose severe problems for cells. Although necessary precursors for thymine nucleotide biosynthesis, deoxyuridine nucleotides can interfere with DNA polymerase activity, either by inhibiting polymerases or by causing them to misincorporate 2′-deoxyuridine 5′-triphosphate (dUTP) in place of 2′-deoxythymidine 5′-triphosphate (dTTP) (1Slupphaug G. Alseth I. Eftedal I. Volden G. Krokan H.E. Anal. Biochem. 1993; 211: 164-169Google Scholar, 2Poole A. Penny D. Sjoberg B.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 147-151Google Scholar). Ubiquitous DNA glycosylase enzymes remove uracil bases from DNA, which are otherwise formed by spontaneous cytosine deamination. The resulting apyrimidinic sites are cleaved by endonucleases, fragmenting cellular DNA (3Sancar A. Sancar G.B. Annu. Rev. Biochem. 1988; 57: 29-67Google Scholar). At temperatures of 70–95 °C, rate constants for spontaneous cytosine deamination (in denatured DNA or cytidine nucleotides) are several orders of magnitude higher than those measured at more moderate temperatures (4Lindahl T. Nyberg B. Biochemistry. 1974; 13: 3405-3410Google Scholar). Therefore hyperthermophilic archaea that grow at such temperatures counter the mutagenic effects of cytosine deamination with a variety of thermostable uracil-DNA glycosylase enzymes (5Sartori A.A. Schar P. Fitz-Gibbon S. Miller J.H. Jiricny J. J. Biol. Chem. 2001; 276: 29979-29986Google Scholar). As a consequence of having efficient base excision repair mechanisms, these organisms must avoid misincorporating deoxyuridine nucleotides into DNA during replication by using highly discriminatory DNA polymerases and by maintaining low dUTP levels in the cell (1Slupphaug G. Alseth I. Eftedal I. Volden G. Krokan H.E. Anal. Biochem. 1993; 211: 164-169Google Scholar, 6Hogrefe H.H. Hansen C.J. Scott B.R. Nielson K.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 596-601Google Scholar). Eukaryotes, some bacteria and some archaea use zinc-dependent cytidine or deoxycytidine deaminase enzymes to produce uridine or deoxyuridine nucleotides. Yet many bacteria, including Escherichia coli and Salmonella typhimurium, produce most of their 2′-deoxyuridine 5′-monophosphate (dUMP) from 2′-deoxycytidine 5′-triphosphate (dCTP) using two different enzymes. These bacteria use dCTP deaminase (DCD,1 EC 3.5.4.13) to catalyze the nucleophilic substitution of a hydroxide ion for the 4-amino group of the cytosine base (7Neuhard J. J. Biol. Chem. 1968; 96: 1519-1527Google Scholar). A second enzyme, dUTP diphosphatase (DUT, EC 3.6.1.23), catalyzes the hydrolysis of the dUTP α-phosphorus anhydride bond to produce pyrophosphate (diphosphate) and dUMP, the obligatory precursor for thymidine nucleotides synthesis (8Shlomai J. Kornberg A. J. Biol. Chem. 1978; 253: 3305-3312Google Scholar) (Fig. 1). Due to the toxicity of dUTP produced by DCD, the DUT enzyme is required to remove the accumulated product. Null mutations in the E. coli dut gene are lethal unless suppressed by null mutations in the dcd gene (9Wang L. Weiss B. J. Bacteriol. 1992; 174: 5647-5653Google Scholar). Despite catalyzing markedly different hydrolytic reactions, DUT and DCD proteins are encoded by homologous genes (9Wang L. Weiss B. J. Bacteriol. 1992; 174: 5647-5653Google Scholar, 10Prangishvili W. Klenk H.-P. Jakobs G. Schmiechen A. Hanselmann C. Holz I. Zillig W. J. Biol. Chem. 1998; 273: 6024-6029Google Scholar). dUTP diphosphatase enzymes have been extensively studied, and four crystal structure models have been published for DUT proteins from E. coli(11Larsson G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Google Scholar), human (12Mol C.D. Harris J.M. Mclntosh E.M. Tainer J.A. Structure. 1996; 4: 1077-1092Google Scholar), and two retroviruses (13Prasad G.S. Stura E.A. McRee D.E. Laco G.S. Hasselkus-Light C. Elder J.H. Stout C.D. Protein Sci. 1996; 5: 2429-2437Google Scholar, 14Dauter Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Google Scholar). However, only one DCD enzyme from S. typhimurium has been partially purified and characterized (15Beck C.F. Eisenhardt A.R. Neuhard J. J. Biol. Chem. 1975; 250: 609-616Google Scholar) and the dcd gene was subsequently identified in E. coli (9Wang L. Weiss B. J. Bacteriol. 1992; 174: 5647-5653Google Scholar). DUT proteins share five conserved signature motifs with the DCD proteins; four of these correspond to conserved uridine binding regions in DUT. These conserved domains have been used to identify dUTP diphosphatase homologs from diverse organisms (16McGeoch D.J. Nucleic Acids Res. 1990; 18: 4105-4110Google Scholar) and to predict that two paralogous genes in the hyperthermophilic archaeonMethanococcus jannaschii encode DCD and DUT proteins (10Prangishvili W. Klenk H.-P. Jakobs G. Schmiechen A. Hanselmann C. Holz I. Zillig W. J. Biol. Chem. 1998; 273: 6024-6029Google Scholar). The original annotation of the complete genome sequence from that organism described both gene products as dCTP deaminases because of their considerable sequence divergence from canonical DUT proteins (17Bult C.J. White O. Olsen G.J. Zhou L. Fleischman R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Doughterty B.A. Tomb J.F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S. Venter J.C. Science. 1996; 273: 1058-1073Google Scholar). In this work, we describe the characterization of two paralogous genes from M. jannaschii that are members of thedut-dcd family. Both genes were cloned inE. coli, and the heterologously expressed proteins were purified and characterized. The protein encoded by locus MJ1102 was shown to be a dUTP diphosphatase, similar to previously characterized DUT homologs. Unexpectedly, the enzyme encoded by locus MJ0430 was found to be a dCTP deaminase/diphosphatase (MjDCD-DUT). It has two functions, catalyzing both the deamination of dCTP and release of pyrophosphate to produce dUMP. It also acts as a dUTP diphosphatase with a lower affinity for dUTP than for dCTP. Compared with theS. typhimurium dCTP deaminase, MjDCD-DUT has a higher affinity for dCTP and is a more active deaminase. The discovery of this bifunctional enzyme ties together the apparently unrelated deaminase and diphosphatase activities of DCD and DUT homologs and shows howM. jannaschii circumvents the problem of accumulating dUTP during thymidine biosynthesis. All reagents were purchased from Sigma unless otherwise specified. Stock solutions of nucleotides were prepared in deionized water. Nucleotide concentrations were determined in sodium phosphate buffer (pH 7.2, 0.1 by using for cytidine uridine and nucleotides for Scholar). The M. jannaschii genes at MJ0430 and MJ1102 were by from DNA using by The MJ0430 gene was using and The MJ1102 gene was using and The at the of the DNA a at the were as described previously using a for MJ0430 and a for MJ1102 M. White J. Bacteriol. Scholar). products were purified using a and with and enzymes DNA was used to the MJ0430 and MJ1102 genes into sites in or S. Proc. Natl. Acad. Sci. U. S. A. Scholar). DNA were by at the of DNA The resulting were coli cells. E. coli were in with were at they at of protein was with with at °C, the were by and at of the proteins was by of cellular the of conserved and of was by and was by The was used to the with in The used were and DNA of the genes were by at the of DNA expressed MjDCD-DUT protein was purified from by and E. coli were in of buffer and in a using a by a at by were for with was at for at The E. coli proteins were denatured by the at for by at for at to remove the cell was to a with buffer A to the were by a protein was with a from to in buffer A at a rate of dCTP deaminase activity were and in a cell with a protein was to a with buffer MjDCD-DUT protein was at a rate of and was in dCTP deaminase activity were and in a cell with a and at expressed protein and the MjDCD-DUT and MjDCD-DUT proteins were purified by the same Protein was by of proteins by using a buffer of the denatured proteins were determined by to low protein Protein concentrations were measured using the protein with as the was at on a The was in buffer and with a rate of Protein used to the were and protein was by their at dCTP deaminase MjDCD-DUT protein was by A of acid was prepared in with MjDCD-DUT of a protein was with of on a were with of acid and a of were in a on the at were by the were identified using the of and used for dCTP deaminase activity was measured using a (15Beck C.F. Eisenhardt A.R. Neuhard J. J. Biol. Chem. 1975; 250: 609-616Google Scholar). In a the reaction was at for buffer (pH 0.1 and enzyme The reaction was by of dCTP to a of The in as a of deamination of cytosine was measured at with The deoxycytidine and deoxyuridine is (15Beck C.F. Eisenhardt A.R. Neuhard J. J. Biol. Chem. 1975; 250: 609-616Google Scholar). dUTP diphosphatase activity buffer (pH 0.1 and enzyme in The reaction was at for the of dUTP. of reaction was by the of of and on from dUTP was measured using in a pyrophosphate phosphate was produced from pyrophosphate by including a thermostable enzyme in the dUTP diphosphatase The enzyme used was heterologously expressed from M. which has pyrophosphate activity, has on nucleotides D.E. White Biochemistry. Scholar). phosphate was by the D.E. White Biochemistry. Scholar). phosphate concentrations were from a using of in deionized from enzyme were from of the to The nucleotide products of the reaction were by on a nucleotides were with a from to in a buffer (pH at a rate of The nucleotides were at Nucleotide were on the and the for The nucleotides their were dUMP dCTP and dUTP of buffer (pH and were of and of a of the MjDCD-DUT The was at for of 0.1 dCTP was at the was to with a of and the was at with of a of and of the by electron impact-mass spectrometry of the was using a at with a from to at The was at the purified MjDCD-DUT was for at temperatures from to and for dCTP deaminase activity using the at The of on dCTP deaminase activity of MjDCD-DUT was using a buffer in the Biochemistry. 1993; Scholar). and were to using either or of and were in dCTP deamination was using enzyme, and dCTP of by a of D.E. White Biochemistry. Scholar, Biochemistry. 2001; Scholar). MjDCD-DUT was by its at concentrations of T. T. or The of MjDCD-DUT was using a Nucleotide at and concentrations and The products of reaction were identified by the as described effects of nucleotides on MjDCD-DUT dCTP deaminase activity were also activity was measured by the of pyrophosphate. These dCTP and concentrations of nucleotides. both dCTP deaminase and dUTP diphosphatase activities of reaction were measured in at concentrations of dCTP or dUTP were at for and were by the of A from the was the rate by using the Sigma dCTP diphosphatase homologs have been identified in the of several archaea (10Prangishvili W. Klenk H.-P. Jakobs G. Schmiechen A. Hanselmann C. Holz I. Zillig W. J. Biol. Chem. 1998; 273: 6024-6029Google Scholar). MJ0430 and MJ1102, were found in the complete genome of M. jannaschii (17Bult C.J. White O. Olsen G.J. Zhou L. Fleischman R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Doughterty B.A. Tomb J.F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S. Venter J.C. Science. 1996; 273: 1058-1073Google Scholar). However, was one gene as a dUTP diphosphatase and the as a dCTP deaminase. cloned both genes and expressed both proteins in E. coli for and The MjDCD-DUT by was purified and shows the of of MjDCD-DUT from of E. coli cells. The activity of purified MjDCD-DUT was activity the which the MjDCD-DUT of the E. coli of M. jannaschii was purified from of E. coli cell was measured using the protein activity was measured with the dCTP deamination MjDCD-DUT was purified from of E. coli cell Protein was measured using the protein activity was measured with the dCTP deamination in a of the purified MjDCD-DUT by spectrometry a of which is to its from the The denatured protein on of a MjDCD-DUT with of that MjDCD-DUT protein a MjDCD-DUT was with dCTP at °C, the enzyme only the deamination of also the release of pyrophosphate to dUMP. MjDCD-DUT can also catalyze the hydrolysis of dUTP to dUMP and pyrophosphate. These that MjDCD-DUT is catalyzes two to dUMP using dCTP as In dCTP deaminase and dUTP diphosphatase enzymes to catalyze the (15Beck C.F. Eisenhardt A.R. Neuhard J. J. Biol. Chem. 1975; 250: 609-616Google Scholar). can catalyze pyrophosphate release by the nucleophilic substitution by at either the or of nucleotide D.J. J. Biol. Chem. 1992; Scholar). the of the enzyme was with dCTP in and the reaction products were to identified two The to the of of these the same for pyrophosphate and The second identified of the of dUMP The of this ion the of two with A ion from the phosphate of the dUMP only a that one of the on the The was at the of uracil during deamination. These that the phosphate anhydride by of at the of The dCTP deaminase was used to the purified MjDCD-DUT reaction with 0.1 the enzyme its activity, with or of enzyme activity was MjDCD-DUT was in (pH (pH or (pH of and the activity, was in the MjDCD-DUT is a thermostable enzyme. at for MjDCD-DUT of its activity, at of MjDCD-DUT dCTP deaminase activity at activity at As shown in the from to the enzyme more than of its The of MjDCD-DUT was using the deamination with concentrations of cytidine nucleotides. is a very MjDCD-DUT catalyzes its deamination and hydrolysis to dUMP at only of the rate of dCTP deamination. of the or nucleotides was by or or were for was pyrophosphate in these These that MjDCD-DUT is highly for dCTP as its for deamination. The MjDCD-DUT activity was by activity was in dCTP deamination activity was with the of in the of dCTP. activity at the of Compared with its dCTP deaminase activity, dUTP diphosphatase activity of MjDCD-DUT a similar of of on MjDCD-DUT dCTP deamination and dUTP diphosphatase activities are expressed as the of activity to that with Mg2+. of dCTP deamination and dUTP diphosphatase activities were as described activities are expressed as the of activity to that with Mg2+. of dCTP deamination and dUTP diphosphatase activities were as described in a the effects of nucleotides on MjDCD-DUT dCTP deaminase activity, concentrations of or were to that dCTP. activity was by the of pyrophosphate using the Sigma pyrophosphate these only enzyme The activity was with dUTP is a for MjDCD-DUT diphosphatase activity, the effects of dUTP were using the deamination dUTP the activity by the of for both dCTP deaminase and dUTP diphosphatase activities were measured at The for both is a in these the was at more than a the Both of activity were to the rate At °C, of MjDCD-DUT were for dCTP and for both dCTP and dUTP are for MjDCD-DUT diphosphatase activity, a was used to the of these two activities 2001; Scholar). concentrations were to similar activities with dCTP or dUTP The of in the was from dCTP and dUTP to dCTP and dUTP. The activity for pyrophosphate was by the phosphate produced the reaction was to a thermostable pyrophosphate enzyme D.E. White Biochemistry. Scholar). The were with a for at the same and with that dCTP and dUTP at the same sites 2001; Scholar). of the MjDCD-DUT protein sequence with homologous DCD and DUT identified conserved in four motifs that were previously described for dUTP (16McGeoch D.J. Nucleic Acids Res. 1990; 18: 4105-4110Google Scholar). structure models of bacterial and DUT proteins one of these conserved to of of in binding the group of the G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Google Scholar, C.D. Harris J.M. Mclntosh E.M. Tainer J.A. Structure. 1996; 4: 1077-1092Google Scholar, G.S. Stura E.A. McRee D.E. Laco G.S. Hasselkus-Light C. Elder J.H. Stout C.D. Protein Sci. 1996; 5: 2429-2437Google Scholar). The bacterial enzyme also use the to a for nucleophilic on the group G. Svensson L.A. Nyman P.O. Nat. Struct. Biol. 1996; 3: 532-538Google Scholar). The DUT protein use this to Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Google Scholar). this to we used to with MjDCD-DUT dCTP deaminase dUTP diphosphatase The protein of measured by with a of for the the of the In the bacterial cytidine deaminase the of a conserved is to as a release of the group a nucleophilic hydroxide ion the cytosine base L. S. R. J. Mol. Biol. Scholar, S. R. Biochemistry. Scholar, E. Neuhard J. S. Biochemistry. 2002; Scholar). Although MjDCD-DUT has sequence to this zinc-dependent cytidine deaminase, MjDCD-DUT homologs share a conserved this is conserved in DCD in DUT we used to with MjDCD-DUT protein dCTP deaminase activity, dUTP diphosphatase activity to enzyme. The purified MjDCD-DUT also has a higher affinity for dUTP with the MjDCD-DUT is also conserved in the archaeal DUT enzyme that dCTP deaminase activity, these that the is for dCTP deaminase of constants for dCTP deaminases and dUTP and of their typhimurium dCTP deaminase E. coli DUT MjDCD-DUT and dUTP diphosphatase are by one active protein of the rate of reaction on dCTP was The rate of the reaction was at a dCTP coli jannaschii has dCTP diphosphatase and of their typhimurium dCTP deaminase (15Beck C.F. Eisenhardt A.R. Neuhard J. J. Biol. Chem. 1975; 250: 609-616Google Scholar), E. coli DUT G. Nyman P.O. J. J. Biol. Chem. 1996; Scholar), MjDCD-DUT and dUTP diphosphatase are by one active protein The of the rate of reaction on dCTP was The rate of the reaction was at a dCTP MjDCD-DUT has dCTP diphosphatase in a protein a on a with of with its of the the protein with of which that the protein a at °C, hydrolysis of dUTP to dUMP and pyrophosphate. It dCTP deaminase Therefore the MJ1102 gene encodes a dUTP shows the acid of MjDCD-DUT and MJ1102 proteins with from homologous DCD proteins and four previously characterized DUT enzymes. All of the share conserved in five previously sequence motifs (10Prangishvili W. Klenk H.-P. Jakobs G. Schmiechen A. Hanselmann C. Holz I. Zillig W. J. Biol. Chem. 1998; 273: 6024-6029Google Scholar, D.J. Nucleic Acids Res. 1990; 18: 4105-4110Google Scholar). structure models of DUT proteins active is of from motifs or by predict that the MjDCD-DUT protein a similar and active Despite the of a of to DCD DUT homologs (10Prangishvili W. Klenk H.-P. Jakobs G. Schmiechen A. Hanselmann C. Holz I. Zillig W. J. Biol. Chem. 1998; 273: 6024-6029Google Scholar), of those acid are conserved in more diverse (Fig. in the of the DCD homologs DCD from DUT proteins and the to deaminase activity on DCD enzymes. two were to deoxycytidine nucleotides to dUMP for thymidine biosynthesis. and some deoxycytidine nucleotides to and the nucleotide using a zinc-dependent deaminase (15Beck C.F. Eisenhardt A.R. Neuhard J. J. Biol. Chem. 1975; 250: 609-616Google Scholar, J. Biol. Chem. 1993; Scholar). produce the only and dUMP from nucleotide to avoid the many bacteria use the DCD protein to dCTP and the DUT protein to dUTP to dUMP. These organisms DUT proteins with affinity for dUTP to avoid accumulating that M. jannaschii has the problem of dUTP by dCTP deamination to the release of pyrophosphate using the MjDCD-DUT enzyme described of dUTP is similar to the use of enzyme by to catalyze the steps in biosynthesis B. G. D. J. Mol. Biol. 1998; Scholar, B. M. G. Biochem. Scholar). the S. typhimurium DCD MjDCD-DUT activity is by (15Beck C.F. Eisenhardt A.R. Neuhard J. J. Biol. Chem. 1975; 250: 609-616Google Scholar). Although the of this is concentrations the by some DUT proteins are by they and dCTP with lower than dUTP (12Mol C.D. Harris J.M. Mclntosh E.M. Tainer J.A. Structure. 1996; 4: 1077-1092Google Scholar). Therefore members of the nucleotides. a bifunctional enzyme that the dUTP M. jannaschii of the to dUTP produced by spontaneous deamination and nucleotide Therefore that organism also has a dUTP Although M. jannaschii DUT has a higher affinity for dUTP than its affinity is lower than that of the E. coli or human DUT enzymes M. jannaschii DUT has similar in the concentrations of DUT to low dUTP levels during DNA Although the of the hydrolytic has to be to dUTP to dUMP and to to dUMP, that the reaction in the of dCTP dUTP dUMP. for dCTP deaminase and dUTP diphosphatase with the of a that deamination and pyrophosphate release occur at the same binding The that MjDCD-DUT protein has deaminase activity can catalyze the release of pyrophosphate a of both at the same binding site, Both the affinity for dCTP and the turnover of MjDCD-DUT are higher than those of the homologous S. typhimurium dCTP deaminase In to the for the S. typhimurium DCD enzyme, MjDCD-DUT dCTP and the rate of deamination. the both enzymes and the for the enzymes is a the cytidine deaminase L. S. R. J. Mol. Biol. Scholar) or deaminase K.S. Biochemistry. 1993; Scholar, K.S. Science. Scholar), MjDCD-DUT use a hydroxide to of the cytosine The of hydroxide by MjDCD-DUT is the enzyme use either nucleophilic or the as a a from water. The of deamination activity in the MjDCD-DUT protein that this is for deaminase is conserved in DCD in most DUT for DUT proteins (Fig. Therefore is necessary be for deaminase In we found that in of DCD proteins from those in DUT homologs. In DUT crystal structure of this in human and DUT this (in a enzyme the the active (12Mol C.D. Harris J.M. Mclntosh E.M. Tainer J.A. Structure. 1996; 4: 1077-1092Google Scholar, G.S. Protein Sci. 2001; 2: Scholar). studies be to the of this to the MjDCD-DUT the bacterial dCTP deaminase, the MjDCD-DUT enzyme also has dUTP diphosphatase structure models of DUT that of active sites in the is of at the of One conserved to of to in the active It has been that this is also in by J.M. Mclntosh E.M. J. Mol. Biol. 1999; Scholar, G. Nyman P.O. J. J. Biol. Chem. 1996; Scholar). A of DUT shows that the a bond with a group from the structure Z. Persson R. Rosengren A.M. Nyman P.O. Wilson K.S. Cedergren-Zeppezauer E.S. J. Mol. Biol. 1999; 285: 655-673Google Scholar). The protein MjDCD-DUT is to catalyze either the dCTP deaminase or the dUTP diphosphatase from that the protein the same structure as the enzyme, the of to be A of the DUT and DCD homologs that dCTP deaminase activity from a dUTP diphosphatase a gene (10Prangishvili W. Klenk H.-P. Jakobs G. Schmiechen A. Hanselmann C. Holz I. Zillig W. J. Biol. Chem. 1998; 273: 6024-6029Google Scholar). Although both dut genes have been in many of gene which have been by A.M. J. 1999; Scholar). Yet these the evolution of deaminase activity in DCD, from gene a dUTP The bifunctional activity of the MjDCD-DUT protein described illustrates a of enzymes a reaction has into a suprafamily of enzymes that catalyze mechanistically distinct J.A. Annu. Rev. Biochem. 2001; Scholar). for with and for some on