Guanosine Monophosphate Reductase Research Articles

Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme–ligand binary complex formation

Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.

An Enzymatic Palimpsest

An Enzymatic Palimpsest

Crystal Structure of Human Guanosine Monophosphate Reductase 2 (GMPR2) in Complex with GMP

Guanosine monophosphate reductase (GMPR) catalyzes the irreversible and NADPH-dependent reductive deamination of GMP to IMP, and plays a critical role in re-utilization of free intracellular bases and purine nucleosides. Here, we report the first crystal structure of human GMP reductase 2 (hGMPR2) in complex with GMP at 3.0 A resolution. The protein forms a tetramer composed of subunits adopting the ubiquitous (alpha/beta)8 barrel fold. Interestingly, the substrate GMP is bound to hGMPR2 through interactions with Met269, Ser270, Arg286, Ser288, and Gly290; this makes the conformation of the adjacent flexible binding region (residues 268-289) fixed, much like a door on a hinge. Structure comparison and sequence alignment analyses show that the conformation of the active site loop (residues 179-187) is similar to those of hGMPR1 and inosine monophosphate dehydrogenases (IMPDHs). We propose that Cys186 is the potential active site, and that the conformation of the loop (residues 129-133) suggests a preference for the coenzyme NADPH over NADH. This structure provides important information towards understanding the functions of members of the GMPR family.

Mice with targeted disruption of the Dio2 gene have cold-induced overexpression of the uncoupling protein 1 gene but fail to increase brown adipose tissue lipogenesis and adaptive thermogenesis.

The Dio2 gene encodes the type 2 deiodinase (D2) that activates thyroxine (T4) to 3,3',5-triiodothyronine (T3), the disruption of which (Dio2(-/-)) results in brown adipose tissue (BAT)-specific hypothyroidism in an otherwise euthyroid animal. In the present studies, cold exposure increased Dio2(-/-) BAT sympathetic stimulation approximately 10-fold (normal approximately 4-fold); as a result, lipolysis, as well as the mRNA levels of uncoupling protein 1, guanosine monophosphate reductase, and peroxisome proliferator-activated receptor gamma coactivator 1, increased well above the levels detected in the cold-exposed wild-type animals. The sustained Dio2(-/-) BAT adrenergic hyperresponse suppressed the three- to fourfold stimulation of BAT lipogenesis normally seen after 24-48 h in the cold. Pharmacological suppression of lipogenesis with betabeta'-methyl-substituted alpha-omega-dicarboxylic acids of C14-C18 in wild-type animals also impaired adaptive thermogenesis in the BAT. These data constitute the first evidence that reduced adrenergic responsiveness does not limit cold-induced adaptive thermogenesis. Instead, the resulting compensatory hyperadrenergic stimulation prevents the otherwise normal stimulation in BAT lipogenesis during cold exposure, rapidly exhausting the availability of fatty acids. The latter is the preponderant determinant of the impaired adaptive thermogenesis and hypothermia in cold-exposed Dio2(-/-) mice.

Cloning and functional characterization of GMPR2, a novel human guanosine monophosphate reductase, which promotes the monocytic differentiation of HL-60 leukemia cells.

To identify the biological function of a novel molecule which shows high homology with human guanosine monophosphate reductase (GMPR) and is named GMPR2. GMPR2 cDNA was cloned from the cDNA library of human dendritic cells and was characterized by Bioinformatics. The expression pattern of GMPR2 was analyzed by Northern blotting. The enzymatic activity of the purified recombinant GMPR2 protein was determined using a spectrophotometric assay. HL-60 leukemia cells were transfected with GMPR2 and the expression of CD14 and myeloperoxidase (MPO) in HL-60 cells with and without 12- o-tetra-decanoyl-phorbol-13-acetate (TPA) induction was monitored by FACS analysis. The novel gene contained ten exons and nine introns and was mapped to 14q11-21. Northern blotting indicated a ubiquitous expression of GMPR2 mRNA in most of the human tissues and cancer cell lines investigated. The recombinant GMPR2 protein was able to reduce GMP. The expression of CD14 and MPO in HL-60 leukemia cells overexpressing GMPR2 clearly increased after induction by TPA. GMPR2 is a novel human GMP reductase, and overexpression of GMPR2 can promote the monocytic differentiation of HL-60 leukemia cells.

The Guanosine Monophosphate Reductase Gene Is Conserved in Rats and Its Expression Increases Rapidly in Brown Adipose Tissue during Cold Exposure

Non-shivering thermogenesis is required for survival of rodents during cold stress. Uncoupling protein-1 acts in brown adipose tissue (BAT) to transport protons, thus dissipating the proton gradient across the inner mitochondrial membrane. This permits respiration uncoupled from ATP synthesis. UCP-1 function is inhibited by the binding of purine nucleotides, with GTP/GDP being more potent than ATP/ADP. We used a cDNA subtraction analysis to identify cDNAs rapidly induced by cold exposure. One of these encodes rat guanosine monophosphate reductase (GMP-r). This was surprising in that previous data had suggested that this enzyme was absent in rodents. Rat GMP-r is 96% identical to human GMP-r, and its mRNA is increased 30-fold in BAT within 6 h of cold exposure. The gene is also expressed (but not cold-responsive) in muscle and kidney, but not in white fat. We speculate that the physiological function of the marked increase in BAT GMP-r during cold stress may be to deplete the brown adipocyte of guanine nucleotides, converting them to IMP, thus permitting enhanced UCP-1 function. This is a previously unrecognized regulatory aspect of thermogenesis, an essential physiological response of rodents to cold.

An integrated map of human chromosome 6p23.

The human chromosomal band 6p23 is a Giemsa-negative (light) band that may be expected to be relatively gene rich. The genes for spinocerebellar ataxia type 1 (SCA1), guanosine monophosphate reductase (GMPR), DEK involved in a subtype of acute myeloid leukemia (AML), and the folate-sensitive fragile site FRA6A, have already been mapped to 6p23. Recent linkage data have suggested evidence for a susceptibility locus for schizophrenia in the region. We have constructed a single YAC contig of approximately 100 clones spanning the entire 6p23 band from 6p22.3 to 6p24.1 and covering 7.5-8.5 Mb of DNA. The YAC contig contains 55 markers including genetically mapped STSs, physically mapped STSs, anonymous STSs, anonymous ESTs, and ESTs from the genes mapped to the region. The order of the genetically mapped STSs is consistent with their order in the contig and some of the markers not resolved on the genetic map have been resolved by the YACs. Four of the YACs from 6p23 and covering approximately 3 Mb of DNA have been used to isolate approximately 300 cosmids from a flow-sorted human chromosome 6 cosmid library, which have been organized into pockets. The proposed susceptibility locus for schizophrenia is most closely linked to D6S260, which is located within the YAC contig along with genetic markers < or = 5 cM on either side. Therefore, the presented materials are valuable reagents for characterization of the genomic region implicated in schizophrenia.

Mapping of the Human Guanosine Monophosphate Reductase Gene (GMPR) to Chromosome 6p23 by Fluorescence in Situ Hybridization

Guanosine monophosphate (GMP) reductase (EC 1.6.6.8) catalyzes the NADPH-dependent reductive deamination of guanosine monophosphate to inosine monophosphate: GMP + NADPH + 2H[sup +] [yields] IMP + NH[sub 4][sup +] + NADP[sup +]. The authors have previously cloned a human GMP reductase gene. They report here the precise localization of the human GMP reductase gene (GMPR) on human chromosomes. 2 refs.

Genomic structure and expression of human guanosine monophosphate reductase.

In vitro translation in the rabbit reticulocyte system and transient expression in Cos7 cells were performed to characterize the protein encoded by a chromosome 6-linked human cDNA clone, whose nucleotide sequence is homologous to that of Escherichia coli guanosine monophosphate reductase (GMP reductase) cDNA. The molecular weight of the peptide produced by the cDNA was about 37,000 Dalton, and the protein produced in the Cos7 cells exhibited GMP reductase activity, substantiating that the cDNA is for human GMP reductase. The corresponding genomic clones were obtained from two human genomic libraries. The gene spans about 50 kb and is composed of 9 exons, which encode 345 amino acid residues. Organization of exons and introns was established by DNA sequencing of each exon and splicing junctions. The gene contains two potential SpI binding sites within exon 1, and a functional atypical polyadenylation signal in exon 9.

Identification of common variant alleles of the human guanosine monophosphate reductase gene

Examination of nucleotide sequences of genomic DNA samples obtained from several unrelated Caucasians and orientals revealed the existence of four variant alleles in the chromosome 6-linked quanosine monophosphate reductase locus. The wild-type gene has T at position 42 (counting from A of the chain initiation codon), C at 630, G at 700, and T at 766, i.e., its structure is T(42)-C(630)-G(700)-T(766). The variant gene, T-T-G-T, was found in about 10% of the loci examined. The C-to-T change at 630 was silent and did not induce any amino acid substitution (His at amino acid residue 210), but it created an additional NcoI cleavage site in the variant gene. The frequency of another variant, the T-C-G-A gene, was about 30%. The T-to-A change at 766 caused an amino acid substitution Phe----Ile at amino acid residue 256 in the variant protein. Frequencies of the C-C-G-T variant and the T-C-A-T variant were probably lower than 5% in Caucasians and orientals.

Studies on the mechanism of cytotoxicity of 3-deazaguanosine in human cancer cells.

The mechanism of toxicity of 3-deazaguanosine was studied in a number of human tumor cell lines by determination of the effects of various purine compounds on the growth of the cells in the presence of the drug and by studies of the effects of 3-deazaguanosine on the metabolism of radiolabeled precursors in these cells. The drug was found to be toxic to all of the cell lines tested. The toxicity was reversible with removal of the drug. None of the purine bases tested could restore normal growth after 48 h exposure to 3-deazaguanosine; the bases were more effective in preventing cytotoxicity when added simultaneously with the drug. Metabolic studies indicated decreased synthesis of DNA, variable inhibition of de novo purine synthesis, and complete inhibition of the enzyme guanosine monophosphate reductase by 3-deazaguanosine.

Glutamine and related analogs regulate guanosine monophosphate reductase in Salmonella typhimurium.

The addition of a glutamine analog, 6-diazo-5-oxo-L-norleucine, or an inhibitor of glutamine synthetase, L-methionine-dl-sulfoximine, to the growth media of most Salmonella typhimurium strains resulted in a marked elevation of guanosine monophosphate reductase levels. The elevation caused by either compound required protein synthesis and could be antagonized by exogenous glutamine. In addition, when glutamine auxotrophs were grown in suboptimal concentrations of glutamine, the guanosine monophosphate reductase levels were increased. It is postulated that glutamine or a product of its metabolism may function under normal conditions as a negative regulatory element in the control of guanosine monophosphate reductase and that decreased effective intracellular levels of glutamine result in an increase in the level of the enzyme.

Utilization and interconversion of purines and ribonucleosides in the mosquito Anopheles albimanus Weidemann

1. 1. The salvage pathways of ribonucleotide synthesis in the adult mosquito, Anopheles albimanus, were established by the incorporation of 8- 14C-labelled purines and ribonucleosides into the RNAs. 2. 2. A comparison of enzymatic activities shows that adenine phosphoribosyltransferase is the most active enzyme in purine nucleotide synthesis. 3. 3. Adenosine is presumably deaminated to inosine. 4. Evidence is presented for the existence of inosine kinase. 4. 5. The data show low hypoxanthine-guanine phosphoribosyltransferase activity. For comparison, the activity of this enzyme is considered in the housefly, Mucosa domestica. 5. 6. The transformation of adenine into guanine is reversible, due to detectable activity found in RNA adenine when radioactive guanine was the precursor. 6. 7. This finding establishes the presence of guanosine monophosphate reductase 7. 8. From the results, it was concluded that adenine phosphoribosyltranferase is a metabolically important enzyme in mosquito maturation and that inhibitors of the enzyme may have potential value as insect control agents.

Guanosine monophosphate reductase from Artemia salina: Inhibition by xanthosine monophosphate and activation by diguanosine tetraphosphate.

In the course of studies on the metabolic role of diguanosine tetraphosphate during development of Artemia salina, a guanosine monophosphate (GMP) reductase has been found in partially purified from the 150 000g Artemia cysts supernatant. From Lineweaver-Burk plots, two apparent Km values of 5 and 50 muM were obtained for GMP. Xanthosine monophosphate (XMP) is a very strong inhibitor of the reaction. In the presence of 1.5 muM XMP hyperbolic kinetics are found. Diguanosine tetraphosphate counteracts very effectively the inhibition of the activity by XMP, concomitantly changing to hyperbolic the kinetics of the enzyme, with a unique Km value of about 5 muM. The complex kinetic and the existence of allosteric e-fectors at physiological concentrations, together with our lack of success in resolving two isoenzymes, makes it very likely that GMP reductase presents negative cooperativity towards its substrate. The effect of diguanosine tetraphosphate on the enzyme is very specific; other structural analogues, diadenosine tetraphosphate and diguanosine triphosphate, tested a micromolar concentrations had no detectable effect on the enzyme. Guanosine triphosphate (GTP) (mM) was also able to counteract the inhibition of guanosine monophosphate (GMP) reductase by XMP. The properties of the Artemia GMP reductase are here compared with those of the similar enzyme from calf thymus and Escherchia coli. As a consequence, the regulation of eukaryotic GMP reductase is resulting to be quite different from that of the reductase from prokaryotes.

Erythrocyte adenosine kinase activity in gout

1. 1. Erythrocyte adenosine kinase (AK) (EC 2.7.1.20) and guanosine monophosphate (GMP) reductase (EC 1.6.6.8) were measured in healthy male controls and primary gouty subjects. Adenosine kinase activity in 19 controls and 26 gouty subjects was 0.717 ± 0.176 and 0.615 ± 0.128 nmol/mg protein/h, respectively. The difference was statistically significant ( P < 0.05). GMP reductase activity in 39 controls and 46 gouty subjects was 30.90 ± 6.28 and 33.43 ± 7.97 μmol/mg protein/h, respectively, without statistically significant difference. 2. 2. When gouty patients were divided into two groups according to gouty heredity and/or tophus formation, a group with such clinical traits had a lower adenosine kinase activity when compared with another group having none of these clinical features ( P < 0.10). Adenosine kinase activity had negative correlations to serum urate level ( r = −0.433, P < 0.05) and to erythrocyte adenosine deaminase (ADA) activity ( r = −0.689, P < 0.05). The possible biochemical role of adenosine kinase activity in purine metabolism and in the pathogenesis of primary gout is discussed.

Metabolic purine pathways in the developing ovary of the housefly, Musca domestica

1. 1. A detailed knowledge of the enzymatic pathways of purine biosynthesis and interconversions in houseflies is important in determining if any biochemical differences exist that might allow exploitation in the selection of compounds that can be used in insect control. 2. 2. Accordingly, the present study demonstrates that the housefly ovaries are deficient in hypoxanthine-guanine phosphoribosyltransferase. When (8- 14C) adenine was injected into houseflies prior to sacrifice, the purine was utilized in the formation of ovarian DNA adenine and guanine. 3. 3. The transformation of adenine into guanine is not reversible since no activity was observed in the adenine fraction when radioactive guanine was administered. Adenine phosphoribosyltransferase appears to be the main salvage pathway enzyme of nucleotide synthesis. 4. 4. There was no evidence to indicate the presence of purine nucleoside phosphorylase or guanosine monophosphate reductase. 5. 5. The nucleoside kinases were present since radioactive guanosine was incorporated into guanine of DNA. The animal and bacterial cells have low kinase activity. 6. 6. The data indicate that free purine base analogues are not inhibitors of nucleic acid synthesis and nucleotide formation is necessary for inhibition in the salvage pathway.

Dependence of diaminopurine utilization on the mutational site of purine auxotrophy in Bacillus subtilis. 1. Nutritional experiments.

An attempt was made to explain the puzzling observation that in bacteria 2,6-diaminopurine can replace guanine for guanineless mutants and for xanthineless mutants (both of which can make adenosine monophosphate de novo) but not for nonexacting purine auxotrophs (which cannot make adenosine monophosphate de novo). The analogue failed to inhibit the growth of nonexacting purineless Bacillus subtilis MB-1356 growing on guanine. In fact, growth was somewhat stimulated. This eliminated a possible solution involving the inhibition of guanosine monophosphate reductase by a diaminopurine derivative. Sparing of guanine by diaminopurine was matched by an even greater sparing of adenine. Addition of a small amount of adenine to MB-1356 failed to allow unrestricted growth on diaminopurine, thus eliminating a possible solution requiring an adenine derivative for the initial deamination of diaminopurine to guanine. The same degree of sparing of adenine by diaminopurine was observed whether both purines were added together or whether the adenine was added 1 hr after diaminopurine. This eliminated the possibility that diaminopurine was wasted by a "dead-end" conversion in the absence of adenine. Consideration of these nutritional data led to the development of two additional explanations, which are examined by tracer methodology in the following paper.