Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) from Staphylococcus lugdunensis.

The 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) catalyzes final two steps of purine nucleotide de novo biosynthetic pathway. This study reports the characterization of ATIC from Staphylococcus lugdunensis (SlugATIC). Apart from kinetic analysis and a detailed biophysical characterization of SlugATIC, the role of ATIC in cell proliferation has been demonstrated for the first time. The purified recombinant SlugATIC and its truncated domains exist mainly in dimeric form was revealed in gel-filtration and glutaraldehyde cross-linking studies. The two activities reside on separate domains was demonstrated in kinetic analysis of SlugATIC and reconstituted truncated N-terminal IMP cyclohydrolase (IMPCHase) and C-terminal AICAR transformylase (AICAR TFase) domains. Site-directed mutagenesis showed that Lys255 and His256 are the key catalytic residues, while Asn415 substantially contributes to AICAR TFase activity in SlugATIC. The differential scanning calorimetry (DSC) analysis revealed a molten globule-like structure for independent N-terminal domain as compared with a relatively stable conformational state in full-length SlugATIC signifying the importance of covalently linked domains. Unlike reported crystal structures, the DSC studies revealed significant conformational changes on binding of leading ligand to AICAR TFase domain in SlugATIC. The cell proliferation activity of SlugATIC was observed where it promoted proliferation and viability of NIH 3T3 and RIN-5F cells, exhibited in vitro wound healing in NIH 3T3 fibroblast cells, and rescued RIN-5F cells from the cytotoxic effects of palmitic acid and high glucose. The results suggest that ATIC, an important drug target, can also be exploited for its cell proliferative properties.

Mechanism of dTTP inhibition of the bifunctional dCTP deaminase:dUTPase encoded by Mycobacterium tuberculosis.

Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be characterised and provides evidence for bifunctionality of dCTP deaminase occurring outside the Archaea kingdom. A steady-state kinetic analysis revealed that the affinity for dCTP and deoxyuridine triphosphate as substrates for the synthesis of deoxyuridine monophosphate were very similar, a result that contrasts that obtained previously for the archaean Methanocaldococcus jannaschii enzyme, which showed approximately 10-fold lower affinity for deoxyuridine triphosphate than for dCTP. The crystal structures of the enzyme in complex with the inhibitor, thymidine triphosphate, and the apo form have been solved. Comparison of the two shows that upon binding of thymidine triphosphate, the disordered C-terminal arranges as a lid covering the active site, and the enzyme adapts an inactive conformation as a result of structural changes in the active site. In the inactive conformation dephosphorylation cannot take place due to the absence of a water molecule otherwise hydrogen-bonded to O2 of the alpha-phosphate.

A novel function for the N-terminal nucleophile hydrolase fold demonstrated by the structure of an archaeal inosine monophosphate cyclohydrolase.

Inosine 5'-monophosphate (IMP) cyclohydrolase catalyzes the cyclization of 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) to IMP in the final step of de novo purine biosynthesis. Two major types of this enzyme have been discovered to date: PurH in Bacteria and Eukarya and PurO in Archaea. The structure of the MTH1020 gene product from Methanothermobacter thermoautotrophicus was previously solved without functional annotation but shows high amino acid sequence similarity to other PurOs. We determined the crystal structure of the MTH1020 gene product in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) at 2.0 and 2.6 A resolution, respectively. On the basis of the sequence analysis, ligand-bound structures, and biochemical data, MTH1020 is confirmed as an archaeal IMP cyclohydrolase, thus designated as MthPurO. MthPurO has a four-layered alphabeta betaalpha core structure, showing an N-terminal nucleophile (NTN) hydrolase fold. The active site is located at the deep pocket between two central beta-sheets and contains residues strictly conserved within PurOs. Comparisons of the two types of IMP cyclohydrolase, PurO and PurH, revealed that there are no similarities in sequence, structure, or the active site architecture, suggesting that they are evolutionarily not related to each other. The MjR31K mutant of PurO from Methanocaldococcus jannaschii showed 76% decreased activity and the MjE102Q mutation completely abolished enzymatic activity, suggesting that these highly conserved residues play critical roles in catalysis. Interestingly, green fluorescent protein (GFP), which has no structural homology to either PurO or PurH but catalyzes a similar intramolecular cyclohydrolase reaction required for chromophore maturation, utilizes Arg96 and Glu222 in a mechanism analogous to that of PurO.

A bifunctional dCTP deaminase-dUTP nucleotidohydrolase from the hyperthermophilic archaeon Methanocaldococcus jannaschii.

By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the alpha- and beta-phosphates. When the reaction was followed by thin layer chromatography using [3H]dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from [3H]dCTP to the pool of dUTP. This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme. The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released. The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (Km >100 micro m). According to limited proteolysis, the C-terminal residues constitute a flexible region. The other protein investigated, MJ1102, is a specific dUTPase with a Km for dUTP (0.4 micro m) comparable in magnitude with that found for previously characterized dUTPases. Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism.

The kinetic mechanism of the human bifunctional enzyme ATIC (5-amino-4-imidazolecarboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase). A surprising lack of substrate channeling.

5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of substrate channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.

New class of IMP cyclohydrolases in Methanococcus jannaschii.

The enzyme responsible for observed IMP cyclohydrolase activity in Methanococcus jannaschii was purified and sequenced: its genetic locus was found to correspond to gene MJ0626. The MJ0626 gene was cloned, and its protein product was expressed in Escherichia coli and shown to catalyze the cyclization of 5-formylamidoimidazole-4-carboxamide ribonucleotide to IMP. The enzyme has no sequence similarity to known enzymes, and its catalytic properties appear distinct from any characterized IMP cyclohydrolase. The purO gene for the enzyme is currently found only in the domain Archaea.

Biosynthesis of riboflavin: characterization of the bifunctional deaminase-reductase of Escherichia coli and Bacillus subtilis.

The ribG gene at the 5' end of the riboflavin operon of Bacillus subtilis and a reading frame at 442 kb on the Escherichia coli chromosome (subsequently designated ribD) show similarity with deoxycytidylate deaminase and with the RIB7 gene of Saccharomyces cerevisiae. The ribG gene of B. subtilis and the ribD gene of E. coli were expressed in recombinant E. coli strains and were shown to code for bifunctional proteins catalyzing the second and third steps in the biosynthesis of riboflavin, i.e., the deamination of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (deaminase) and the subsequent reduction of the ribosyl side chain (reductase). The recombinant proteins specified by the ribD gene of E. coli and the ribG gene of B. subtilis were purified to homogeneity. NADH as well as NADPH can be used as a cosubstrate for the reductase of both microorganisms under study. Expression of the N-terminal or C-terminal part of the RibG protein yielded proteins with deaminase or reductase activity, respectively; however, the truncated proteins were rather unstable.

5-Aminoimidazole-4-carboxamide ribotide transformylase-IMP cyclohydrolase from human CCRF-CEM leukemia cells: purification, pH dependence, and inhibitors.

The bifunctional enzyme 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase-IMP cyclohydrolase has been purified 780-fold to apparent homogeneity from human CCRF-CEM leukemia cells, completed with chromatography on Affi-Gel Blue followed by AICAR-Sepharose 4B. Using a sensitive radioassay, IMP cyclohydrolase has a Ks value for 5-formamidoimidazole-4-carboxamide ribotide (FAICAR) at pH 7.4 of 0.87 +/- 0.11 microM. The following purine nucleotide derivatives were potent competitive inhibitors of IMP cyclohydrolase: 2-mercaptoinosine 5'-monophosphate (Ki = 0.094 +/- 0.024 microM), xanthosine 5'-monophosphate (Ki = 0.12 +/- 0.01 microM), 2-fluoroadenine arabinoside 5'-monophosphate (Ki = 0.16 +/- 0.02 microM), 6-mercaptopurine riboside 5'-monophosphate (Ki = 0.20 +/- 0.02 microM), adenosine N1-oxide 5'-monophosphate (Ki = 0.28 +/- 0.03 microM), and N6-(carboxymethyl)adenosine 5'-monophosphate (Ki = 1.7 +/- 0.42 microM). The pH dependencies of Vmax and Vmax/Ks values for IMP cyclohydrolase are consistent with a single ionizable amino acid residue (pKa = 7.57 +/- 0.09) of the enzyme which must be unprotonated for catalysis to occur and a residue (pKa = 7.57 +/- 0.14) which must be unprotonated for FAICAR to bind. The pKa values of 5.81 +/- 0.03 and 9.41 +/- 0.04 determined for FAICAR indicate that ionization of the substrate does not contribute significantly to the pH effects observed. Chemical modification of IMP cyclohydrolase provides evidence for arginine and cysteine residues at the active site, and roles for these residues in the mechanism of catalysis are proposed.

Purification and properties of adenosine 5'-phosphosulfate deaminating enzyme from marine macroalga Gloiopeltis furcata.

An enzyme which catalyzed the hydrolytic removal of the 6-amino group of adenosine 5'-phosphosulfate (APS) into inosine 5'-phosphosulfate was purified from the marine red macroalga Gloiopeltis furcata by means of salt fractionation, affinity, anion-exchange, and hydrophobic interaction chromatographies. The native enzyme had a Mr of about 285,000. Dissociation yielded a form with a Mr of about 70,000. The enzyme catalyzed the irreversible deamination of adenosine and its 5'-substituted compounds in addition to APS. Thus the enzyme seemed to be a nonspecific adenine nucleotide deaminase. Some properties were determined and compared with those of other nonspecific adenine nucleotide deaminases.

The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes.

AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.

[Subunit structure of AMP-deaminase from skeletal muscles]. / Sub''edinichnoe stroenie AMP-dezaminazy skeletnykh myshts.

AMP-deaminase was purified from skeletal muscle of rat by the affinity chromatography on phosphocellulose and gel-filtration on Sephadex G-200. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) has shown three protein bands on each step of purification. One of them corresponds to the subunit of tetrameric AMP-deaminase molecule with molecular weight of 76 kDa and two others--to the protein subunit with molecular weight of 42 and 33 kDa. Repeated SDS-PAGE of the main subunit band has revealed again all these protein bands. The data obtained indicate that AMP-deaminase subunit of 76 kDa is able to dissociate on two polypeptide chains with similar values of molecular weights in the presence of SDS.

Dimeric structure of rat skeletal muscle AMP-deaminase subunit.

AMP-deaminase from rat skeletal muscle was purified by affinity chromatography on phosphocellulose and gel-filtration on Sephadex G-200. It was established that disulfide bridges and hydrogen bonds were not essential for stability of enzyme oligomeric structure. The dimeric structure of enzyme subunit with Mr 76 kDa (S1) was detected by means of PAGE in the presence of SDS: besides the S1 there were also exhibited two additional bands with Mr 42 (S2) and 33 (S3) kDa. Repeated SDS-PAGE of S1 has revealed the same three protein bands. These results indicate the possibility of dissociation of S1-subunit into two subunits with close Mr values.

Purification and general properties of AMP deaminase from sheep brain.

AMP deaminase from sheep brain was purified to homogeneity on SDS-PAGE and its general properties were investigated. The native enzyme has a molecular weight of approximately 350,000 as estimated by gel filtration and it is composed of four identical subunits with a molecular weight of 85,000 each. The purified enzyme had a specific activity of 500 units/mg protein and shows a sigmoid-shaped AMP saturation curve in the presence of 100 mM KCl. This deaminase is strongly activated by ATP and inhibited by GTP. It slightly catalyzes the hydrolysis of adenosine monosulfate (AMS), dAMP, and adenosine phosphoramidate (APA). These catalytic properties resemble those of AMP deaminase from human liver.

Enzymes of the purine metabolism in rat brain microsomes.

Rat brain microsomes, when they are suspended in moderate ionic strength medium, released enzyme activities of lactate dehydrogenase (LDH, E.C.1.1.1.27), malate dehydrogenase (MDH, E.C.1.1.1.37), adenosine deaminase (ADA, E.C.3.5.4.4), guanine deaminase (GAH, E.C.3.5.4.3), and purine nucleoside phosphorylase (PNP, E.C.2.1.2.4). The activities released decreased when the saline concentration of the medium was increased and the opposite occurred when 50 mM, pH 7.4 sodium phosphate medium was used. Rat brain microsomes that had been extracted previously by moderate ionic strength solutions still had activities of all the enzymes tested, and released these activities upon sonication or deoxycholate (DOC) treatment. The proportion of the activity released was similar for all the enzymes. DOC treatment released higher enzymic activities and a smaller amount of protein than sonication did. The proportion of activities released was similar to that found in the 105,000 g supernatant. The suspension of microsomes still retained activities of the above-mentioned enzymes after consecutive extractions with increasing concentrations of detergent solutions (DOC and Triton X-100). The amount of enzymic activities released from the microsomes by sonication or DOC treatment did not depend on the protein composition of the homogenization medium. Thus, on increasing the enzyme concentration in the homogenization medium, the activities released did not increase in parallel. The set of results obtained showed that the microsomal fraction is as useful as the cytosolic one for studying purine catabolism in rat brain. Furthermore, the conditions in which purine enzymes are attached to the microsomal fraction are probably closer to "in vivo" conditions than those in which these enzymes are found in the soluble fraction.

AMP deaminase from the gill of the mullet Chelon labrosus R.: purification and effects of pH, phosphate and monovalent cations.

The AMP deaminase from the gill of Chelon labrosus was purified about 250-fold by chromatography on cellulose-phosphate. At low and high substrate concentration, a sharp pH optimum was found between pH 6.7 and 6.9. The enzyme was found to be relatively insensitive to physiological concentrations of inorganic phosphate. Na+ and K+ were activators of gill AMP deaminase, Na+ being the most efficient. Effects of possible changes in the intracellular concentrations of these cations on enzyme activity are discussed.

[Partial purification and properties of the adenylate deaminase from subfractions of soluble mitochondrial proteins of rat liver]. / Chastichnaia ochistka i svoistva adenilatdezaminazy iz subfraktsii rastvorimykh mitokhondrial'nykh belkov pecheni krys.

Isolation and partial purification of AMP-deaminase from subfraction of soluble proteins of the mitochondrial fraction from rat liver is described. The enzyme preparations obtained deaminated AMP at the highest rate from pH 6.4 to 6.6. At the optimal pH value and in presence of optimal AMP concentrations the AMP-deaminase preparation was not activated by ATP or K+ and was inhibited by inorganic phosphate. Relationship was noted between both the content of protein in the enzyme preparations and length of the interval from composing the samples to monitoring the enzymatic activity and the following parameters of the AMP-deaminase: (a) shape of curves describing the rate of AMP deamination as a function of the nucleotide concentration, (b) reversible decrease in the AMP-deaminating activity after dialysis, (c) properties to deaminate, besides AMP, also some other nucleotides (ADP, NAD, FAD), (d) dynamics of inactivation of the enzyme preparations by controlled heating. The properties of the partially purified AMP-deaminase from the subfraction of rat liver soluble mitochondrial proteins were not identical with those described previously for other AMP-deaminases.

[Purification and some physico-chemical properties of myocardial adenylate deaminase]. / Ochistka i nekotorye fiziko-khimicheskie svoistva Adenilatdezaminazy miokarda.

A procedure for isolation of adenylate deaminase from duck heart muscle has been developed. The method includes extraction of enzyme, chromatography on cellulose phosphate, fractionation by ammonium sulfate, chromatography on Sephadex G-25 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified approximately 4000-fold with a yield of 25%. Electrophoresis in polyacrylamide gel revealed that the enzyme contains no proteins other than adenylate deaminase. The enzyme has a UV absorption spectrum typical for proteins which contain no nucleic acid impurities. Using sievorptive chromatography, it was shown that the myocardial extract contains two adenylate deaminase forms, which are tetramers with mol. weights of 190 000 and 240 000. The molecular weights of the subunits are 47 000 and 63 000, respectively. In the oligomeric form the enzyme is only detected at high enzyme concentrations and in the presence of large amounts of substrate.