Cytidine deaminase from Escherichia coli B. Purification and enzymatic and molecular properties

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from Escherichia coli has been purified to homogeneity through a rapid and efficient two-step procedure consisting of anion-exchange chromatography followed by preparative electrophoresis. The final preparation is homogeneous, as judged by a single band obtained by disc gel electrophoresis performed in the absence and presence of denaturing agents. The native protein molecular weight determined by gel filtration is 56 000. Sodium dodecyl sulfate disc gel electrophoresis experiments conducted upon previous incubation of the enzyme with dimethyl suberimidate suggest an oligomeric structure of two identical subunits of 33 000 molecular weight. The absorption spectrum of the protein reveals a maximum at 277 nm and a minimum at 255 nm. The isoelectric point is at pH 4.35. Amino acid analysis indicates an excess of acidic amino acid residues as well as six half-cystine residues. No interchain disulfide groups have been evidenced. According to Cleland's nomenclature, kinetic analysis shows a rapid-equilibrium random Uni-Bi mechanism. Cytidine deaminase is competitively inhibited by various nucleosides. Km values for cytidine, deoxycytidine, and 5-methylcytidine are 1.8 X 10(-4), 0.9 X 10(-4), and 12.5 X 10(-4) M, respectively.     

Purification of cytidine deaminase from chicken liver

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from chicken liver has been obtained in pure form through a rapid procedure consisting of organic solvent precipitation, heat treatment, anionic-exchange chromatography, hydrophobic interaction chromatography followed by two rapid chromatographies using an FPLC system. The final preparation is pure but shows microheterogeneity as judged by a single band obtained by SDS-gel electrophoresis and a series of superimposed active bands obtained on native gel electrophoresis and gel isoelectrofocusing. The native protein molecular weight determined by gel filtration is 50,000. SDS-gel electrophoresis experiments conducted in the presence and in the absence of reducing agents, suggest an oligomeric structure of four apparently identical subunits of 12,000 molecular weight. The absorption spectrum of the native protein reveals a maximum at 278 nm and a minimum at 261 nm with a small shoulder at 285 nm. The isoelectric point has an average value around pH 4.45.     

Characteristics of cytidine aminohydrolase activity in Trypanosoma cruzi and Crithidia fasciculata

Cytidine deaminase (cytidine aminohydrolase, 3.5.4.5) is present in Crithidia fasciculata (a mosquito parasite) and in Trypanosoma cruzi (a human pathogen). The enzyme from C. fasciculata deaminated both cytidine and deoxycytidine, the affinity for the former being much lower than the latter. Affinities for both substrates are equal for the T. cruzi enzyme. The production of the enzyme in C. fasciculata was significantly stimulated by the addition of a number of pyrimidine nucleosides (cytidine, uridine, 5-bromouridine, thymidine, orotidine) to the culture media. Only cytidine stimulated enzyme production in T. cruzi. The enzyme from both organisms was unstable in air, even in the frozen state. Stabilization was achieved under anaerobic conditions.     

Induction of pyrimidine nucleoside metabolizing enzymes in E. coli B

Induction studies on pyrimidine metabolizing enzymes in E. coli B have shown that the enzymes fall into three distinct groups according to their induction pattern. a) Cytidine deaminase and uridine phosphorylase, are induced by cytidine, CMP and adenosine; no induction was observed with uridine and AMP; b) thymidine phosphorylase is induced by cytidine, adenosine, all deoxyribonucleosides, CMP, deoxyribonucleotides, deoxyribose and deoxyribose-1-phosphate; c) uridine-cytidine kinase, uracil phosphoribosyltransferase, 5'-nucleotidase, thymidine kinase, are uninducible enzymes. Simultaneous addition of cytidine and glucose partially overcomes the cytidine deaminase and uridine phosphorylase induction. Cytidine deaminase reaches its maximum activity levels, in E. coli growing cells in presence of cytidine, two hours before the uridine phosphorylase activity. Maximum glucose repression of cytidine deaminase and uridine phosphorylase was obtained in correspondence of maximum cytidine induction.     

Characteristics of Cytidine Aminohydrolase Activity in Trypanosoma cruzi and Crithidia fasciculata1

Cytidine deaminase (cytidine aminohydrolase, 3.5.4.5) is present in Crithidia fasciculata (a mosquito parasite) and in Trypanosoma cruzi (a human pathogen). The enzyme from C. fasciculata deaminated both cytidine and deoxycytidine, the affinity for the former being much lower than the latter. Affinities for both substrates are equal for the T. cruzi enzyme. The production of the enzyme in C. fasciculata was significantly stimulated by the addition of a number of pyrimidine nucleosides (cytidine, uridine, 5-bromouridine, thymidine, orotidine) to the culture media. Only cytidine stimulated enzyme production in T. cruzi. The enzyme from both organisms was unstable in air, even in the frozen state. Stabilization was achieved under anaerobic conditions.     

Purification of human cytidine deaminase: molecular and enzymatic characterization and inhibition by synthetic pyrimidine analogs

Cytidine deaminase has been purified to homogeneity from human placenta by a rapid and efficient procedure consisting of affinity chromatography followed by hydrophobic interaction chromatography. The final enzyme preparation showed a specific activity of 64.1 units/mg, corresponding to about 46,000-fold purification with respect to the crude extract. The enzyme is a 52-kDa oligomeric protein composed of four apparently identical subunits. The acidic isoelectric point is 4.5. The enzyme's stability is strictly dependent on the presence of reducing agents. Amino acid analysis reveals the presence of five thiol groups per monomer which cannot be titrated by Ellman's reagent in the native enzyme. However, the presence of sulfhydryl groups involved in the catalytic activity was evidenced by the inhibition exerted by p-chloromercuribenzoate and heavy metal ions. In addition, the protection effected by the substrate against the p-chloromercuribenzoate inhibition and the competitive inhibition exerted by 5-(chloromercuri)cytidine suggest the presence of a thiol group(s) in the catalytic site of the enzyme. pH studies have shown that the rapid decline of activity occurring at pH 4.5 might result from the protonation of the pyrimidine ring at the N-3 position. The enzyme catalyzes the deamination of cytidine, deoxycytidine, and several analogs, including antineoplastic agents, thus abolishing their pharmacological activity. Therefore, several pyrimidine nucleoside analogs have been tested as potential inhibitors of the enzyme. The competitive inhibition exerted by cytidine analogs having the ribose moiety replaced by aliphatic chains is interesting.     

Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s?1 at 30 °C. Since adenine is deaminated ～103 slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.     

The role of zinc in Bacillus subtilis cytidine deaminase

Cytidine deaminase (CDA) from Bacillus subtilis is a zinc-containing enzyme responsible for the hydrolytic deamination of cytidine to uridine and 2'-deoxycytidine to 2'-deoxyuridine. Titration of the cysteinyl groups of the enzyme with p-hydroxymercuriphenyl sulfonate (PMPS) resulted in release of one zinc ion per subunit. Addition of EDTA to chelate the zinc and dithiothreitol (DTT) to remove PMPS, followed by removal of the low molecular weight compounds by gel filtration, resulted in an apoenzyme with no enzymatic activity. The apoenzyme was almost fully reactivated by addition of zinc chloride, indicating that the zinc ion played a central role in catalysis, in keeping with what has been observed with Escherichia coli CDA [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., and Carter, C. W. J. (1994) J. Mol. Biol. 235, 635-656]. Addition of Cd(2+) or Co(2+) caused partial reactivation of the apoenzyme. Zinc reconstitution of the apoenzyme was strictly dependent on the presence of reducing agents, suggesting that the zinc-ligating cysteines, when unligated, participated in disulfide bond formation. An enzymatically active isoform of the tetrameric CDA protein, containing an extension of 13 amino acids at the C-terminus of each subunit, was used in conjunction with the wild-type CDA in subunit-subunit dissociation studies to show that the zinc ion does not assist in the thermodynamic refolding of the protein. After treatment with PMPS and EDTA, the enzyme existed as unfolded unassociated subunits. Immediately following DTT addition to remove PMPS, the subunits refolded into a tetrameric structure, independent of the presence of zinc.     

Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana

The complementary DNA (cDNA) coding for Arabidopsis thaliana cytidine deaminase 1 (AT-CDA1) was obtained from the amplified A. thaliana cDNA expression library, provided by R. W. Davis (Stanford University, CA). AT-CDA1 cDNA was subcloned into the expression vector pTrc99-A and the protein, expressed in Escherichia coli following induction with isopropyl 1-thio-beta-d-galactopyranoside, showed high cytidine deaminase activity. The nucleotide sequence showed a 903-bp open reading frame encoding a polypeptide of 301 amino acids with a calculated molecular mass of 32,582. The deduced amino acid sequence of AT-CDA1 showed no transit peptide for targeting to the chloroplast or mitochondria indicating that this form of cytidine deaminase is probably expressed in the cytosol. The recombinant AT-CDA1 was purified to homogeneity by a heat treatment followed by an ion-exchange chromatography. The final enzyme preparation was >98% pure as judged by SDS-PAGE and showed a specific activity of 74 U/mg. The molecular mass of AT-CDA1 estimated by gel filtration was 63 kDa, indicating, in contrast to the other eukaryotic CDAs, that the enzyme is a dimer composed of two identical subunits. Inductively coupled plasma-optical emission spectroscopy analysis indicated that the enzyme contains 1 mol of zinc atom per mole of subunit. The kinetic properties of AT-CDA1 both toward the natural substrates and with analogs indicated that the catalytic mechanism of the plant enzyme is probably very similar to that of the human the E. coli enzymes.     

Advantages and disadvantages of cytidine deamination

Cytidine deamination of nucleic acids underlies diversification of Ig genes and inhibition of retroviral infection, and thus, it would appear to be vital to host defense. The host defense properties of cytidine deamination require two distinct but homologous cytidine deaminases-activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G. Although cytidine deamination has clear benefits, it might well have biological costs. Uncontrolled cytidine deamination might generate misfolded polypeptides, dominant-negative proteins, or mutations in tumor suppressor genes, and thus contribute to tumor formation. How cytidine deaminases target a given nucleic acid substrate at specific sequences is not understood, and what protects cells from uncontrolled mutagenesis is not known. In this paper, I shall review the functions and regulation of activation-induced cytidine deaminase and apolipoprotein B-editing cytidine deaminase, subunit 3G, and speculate about the basis for site specificity vis-à-vis generalized mutagenesis.     

Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens.

The membrane-associated phospholipid biosynthetic enzyme cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase; EC 2.7.8.8) was partially purified 337-fold from a cell-free extract of the gram-positive pathogenic anaerobe Clostridium perfringens (ATCC 3624). The purification procedure included extraction from the cell envelope with the nonionic detergent Triton X-100, followed by affinity chromatography on cytidine 5'-diphosphate-diacylglycerol-Sepharose. When the partially purified enzyme was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two major bands were evident with apparent minimum molecular weights of 39,000 and 31,000. Activity of phosphatidylserine synthase was dependent on the addition of manganese ions (3 mM) and Triton X-100 (2.7 mM) for maximum activity. The rate of catalysis was maximal at 40 degrees C (with rapid thermal inactivation above this temperature), and the pH optimum was 8.5. The apparent Km values for cytidine 5'-diphosphate-diacylglycerol and L-serine were 0.24 and 0.26 mM, respectively. The synthetic (forward) reaction was favored, as indicated by an equilibrium constant of 82, and the energy of activation was found to be 18 kcal/mol (75,362 J/mol).     

Cytidine deaminases from B. subtilis and E. coli: compensating effects of changing zinc coordination and quaternary structure.

Cytidine deaminase from E. coli is a dimer of identical subunits (M(r) = 31 540), each containing a single zinc atom. Cytidine deaminase from B. subtilis is a tetramer of identical subunits (M(r) = 14 800). After purification from an overexpressing strain, the enzyme from B. subtilis is found to contain a single atom of zinc per enzyme subunit by flame atomic absorption spectroscopy. Fluorescence titration indicates that each of the four subunits contains a binding site for the transition state analogue inhibitor 5-fluoro-3,4-dihydrouridine. A region of amino acid sequence homology, containing residues that are involved in zinc coordination in the enzyme from E. coli, strongly suggests that in the enzyme from B. subtilis, zinc is coordinated by the thiolate side chains of three cysteine residues (Cys-53, Cys-86, and Cys-89) [Song, B. H., and Neuhard, J. (1989) Mol. Gen. Genet. 216, 462-468]. This pattern of zinc coordination appears to be novel for a hydrolytic enzyme, and might be expected to reduce the reactivity of the active site substantially compared with that of the enzyme from E. coli (His-102, Cys-129, and Cys-132). Instead, the B. subtilis and E. coli enzymes are found to be similar in their activities, and also in their relative binding affinities for a series of structurally related inhibitors with binding affinities that span a range of 6 orders of magnitude. In addition, the apparent pK(a) value of the active site is shifted upward by less than 1 unit. Sequence alignments, together with model building, suggest one possible mechanism of compensation.     

Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDPdiglyceride):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase) is bound tightly to the ribosomes in crude extracts of Escherichia coli. After separation of the enzyme from the ribosomes by the method of Raetz and Kennedy (Raetz, C.R.H., and Kennedy, E.P. (1974), J. Biol. Chem. 249, 5038), we have purified the enzyme to 97% of homogenekty. The major portion of the overall 5500-fold purification was attained by substrate-specific elution from phosphocellulose using CDP-diglyceride in the presence of detergent. The purified enzyme migrated as a single band with an apparent minimum molecular weight of 54 000 when subjected to electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. The purified enzyme catalyzed exchange reactions between cytidine 5'- monophosphate (CMP) and CDP-diglyceride and between serine and phosphatidylserine. The enzyme also catalyzed the hydrolysis of CDP-diglyceride to form CMP and phosphatidic acid. dCDP-diglyceride was equivalent to CDP-diglyceride in all reactions catalyzed by the enzyme. In addition, the purified enzyme catalyzed the formation of phosphatidylglycerol or phosphatidylglycerophosphate at a very slow rate when serine was replaced as substrate by glycerol or sn-glycero-3-phosphate, respectively. These results suggest catalysis occurs via a ping-pong mechanism through the formation of a phosphatidyl-enzyme intermediate.     

Human T-lymphoblast deoxycytidine kinase: purification and properties

Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.     

Genome-Wide Identification and Expression Analysis of the Cytidine Deaminase Subfamily in Rice

Russian Journal of Plant Physiology - Cytidine deaminase (CDA) belongs to the subfamily of cytidine deaminase-like family of proteins, which is involved in the nucleotide metabolism. As CDAs...     

Synthesis and degradation of 1-aminocyclopropane-1-carboxylic acid by Penicillium citrinum

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M(r) 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the Km for S-adenosyl-L-methionine of 1.74 mM and kcat of 0.56 s-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M(r) 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a Km for ACC of 4.8 mM and kcat of 3.52 s-1. The presence of 7 mM Cu2+ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.     

Isolation and various characteristics of tryptophan deaminase from Proteus vulgaris

Tryptophan deaminase was isolated from Proteus vulgaris and purified. The procedure for enzyme purification included the cell destruction on USD-1, fractionation by ammonium sulphate, gel chromatography on ultragel AcA34, ion exchange chromatography on DEAE-cellulose. A degree of the enzyme purification--95, yield--5.7%. The pH optimum was 7.5, the temperature optimum--47 degrees C. The enzyme molecular weight (105 kD) was estimated by gel chromatography on Sephadex G-200, Km--5.0 mM in the K-phosphate buffer (pH 7.5). The SH groups are supposed to be present in the active site of the enzyme. The enzyme does not accelerate oxidation deamination of phenylalanine and tyrosine.     

Binding of pyrimidin-2-one ribonucleoside by cytidine deaminase as the transition-state analogue 3,4-dihydrouridine and the contribution of the 4-hydroxyl group to its binding affinity

Cytidine deaminase, purified to homogeneity from constitutive mutants of Escherichia coli, was found to bind the competitive inhibitors pyrimidin-2-one ribonucleoside (apparent Ki = 3.6 x 10(-7) M) and 5-fluoropyrimidin-2-one ribonucleoside (apparent Ki = 3.5 x 10(-8) M). Enzyme binding resulted in a change of the lambda max of pyrimidin-2-one ribonucleoside from 303 nm for the free species to 239 nm for the bound species. The value for the bound species was identical with that of an oxygen adduct formed by combination of hydroxide ion with 1,3-dimethyl-2-oxopyrimidinium (239 nm), but lower than that of a sulfur adduct formed by combination of the thiolate anion of N-acetylcysteamine with 1,3-dimethyl-2-oxopyrimidinium (259 nm). The results suggest that pyrimidin-2-one ribonucleoside is bound by cytidine deaminase as an oxygen adduct, probably the covalent hydrate 3,4-dihydrouridine, rather than intact or as an adduct involving a thiol group of the enzyme. In dilute solution at 25 degrees C, the equilibrium constant for formation of a single diastereomer of 3,4-dihydrouridine from pyrimidin-2-one ribonucleoside was estimated as approximately 4.7 x 10(-6), from equilibria of dissociation of water, protonation of 1-methylpyrimidin-2-one, and combination of the 1,3-dimethylpyrimidinium cation with the hydroxide ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient and high-fidelity base editor with expanded PAM compatibility for cytidine dinucleotide

Science China Life Sciences - Cytidine base editor (CBE), which is composed of a cytidine deaminase fused to Cas9 nickase, has been widely used to induce C-to-T conversions in a wide range of...     

AMP deaminase from baker's yeast. Kinetic and molecular properties.

The kinetic and molecular properties of AMP deaminase [AMP aminohydrolase, EC 3.5.4.6] purified from baker's yeast (saccharomyces cerevisiae) were investigated. The enzyme was activated by ATP and dATP, but inhibited by Pi and GTP in an allosteric manner. Alkali metal ions and alkaline earth metal ions activated the enzyme to various extent. Kinetic negative cooperativity was observed in the binding of nucleoside triphosphates. Kinetic analysis showed that the number of interaction sites for AMP (substrate) and Pi (inhibitor) is two each per enzyme molecule. The molecular weight of the native enzyme was estimated to be 360,000 by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme gave a single polypeptide band with a molecular weight of 83,000, suggesting that the native enzyme has a tetrameric structure. Baker's yeast AMP deaminase was concluded to consist of two "promoter" units which each consist of two polypeptide chains with identical molecular weight.