Crystal structure of adenine phosphoribosyltransferase from Leishmania tarentolae: potential implications for APRT catalytic mechanism

The three-dimensional structure of Leishmania tarentolae adenine phosphoribosyltransferase (APRT) in complex with adenosine-5-monophosphate (AMP) and a phosphate ion has been solved. Refinement against X-ray diffraction data extending to 2.2-A resolution led to a final crystallographic R factor of 18.3%. Structural comparisons amongst this APRT enzyme and other 'type I' PRTases whose structures have been determined reveal several important features of the PRTases catalytic mechanism. Based on structural superpositions and molecular interaction potential calculations, it was possible to suggest that the PRPP is the first substrate to bind, while the AMP is the last product to leave the active site, in accordance to recent kinetic studies performed with the Leishmania donovani APRT.     

Three-dimensional structure of human adenine phosphoribosyltransferase and its relation to DHA-urolithiasis

In mammals, adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) is present in all tissues and provides the only known mechanism for the metabolic salvage of adenine resulting from the polyamine biosynthesis pathway or from dietary sources. In humans, APRT deficiency results in serious kidney illness such as nephrolithiasis, interstitial nephritis, and chronic renal failure as a result of 2,8-dihydroxyadenine (DHA) precipitation in the renal interstitium. To address the molecular basis of DHA-urolithiasis, the recombinant human APRT was crystallized in complex with adenosine 5'-monophosphate (AMP). Refinement of X-ray diffraction data extended to 2.1 A resolution led to a final crystallographic R(factor) of 13.3% and an R(free) of 17.6%. This structure is composed of nine beta-strands and six alpha-helices, and the active site pocket opens slightly to accommodate the AMP product. The core of APRT is similar to that of other phosphoribosyltransferases (PRTases), although the adenine-binding domain is quite different. Structural comparisons between the human APRT and other "type I" PRTases of known structure revealed several important features of the biochemistry of PRTases. We propose that the residues located at positions corresponding to Leu159 and Ala131 in hAPRT are responsible for the base specificities of type I PRTases. The comparative analysis shown here also provides structural information for the mechanism by which mutations in the human APRT lead to DHA-urolithiasis.     

Male sterility associated with APRT deficiency in Arabidopsis thaliana results from a mutation in the gene APT1

Four mutants of Arabidopsis thaliana that are deficient in adenine phosphoribosyl transferase (APRT) activity have been isolated by selecting for germination of seeds and growth of the plantlets on a medium containing 2,6-diaminopurine (DAP), a toxic analog of adenine. In all mutants, DAP resistance is due to a recessive nuclear mutation at a locus designated apt. The mutants are male sterile due to pollen abortion after meiosis. Furthermore, it has been shown that metabolism of cytokinins is impaired in the mutant BM3, which has the lowest level of APRT activity among the mutants tested. However, three different cDNAs encoding APRT have been isolated in A. thaliana and this raised the question of the nature of the mutation which results in low APRT activity. The mutation was genetically mapped to chromosome I and lies within 6 cM of the phenotypic marker dis2, indicating that the mutation affects the APT1 gene, a result confirmed by sequencing of mutant alleles. The mutation in the allele apt1-3 is located at the 5′ splicing site of the third intron, and eliminates a BstNI restriction site, as verified by Southern blotting and PCR fragment length analysis. Received: 20 February 1997 / Accepted: 28 August 1997     

Mutation at the APRT locus in Friend erythroleukaemia cells 1. Mutation rates and properties of mutants

Spontaneous mutation at the adenine phosphoribosyl transferase (APRT) locus in clone 707 of the Friend cell line was examined. The frequency of cells resistant to 2,6-diaminopurine (DAP) was found to be 2.6 × 10⁻⁵ with a mutation rate of 1.81 × 10⁻⁶ cell⁻¹ generation⁻¹. APRT activities in 9 DAP-resistant clones were found to vary between 0 and 27% the level observed in wild-type cells. It is suggested that clone 707 cells are heterozygous or functionally hemizygous at the APRT locus.     

水稻基因APRT的克隆及其与温敏核雄性不育的关系

在拟南芥中腺嘌呤磷酸核糖转移酶基因(APRT)突变导致植株雄性不育.本文首次报道从水稻(Oryza sativa subsp.indica)中克隆了基因APRT(GenBank登录号AY238894),并将其定位于水稻第4染色体的一个BAC克隆(AL606604)的58 000 bp至63 000 bp区域.该基因长4 220 bp(起始密码子至终止密码子),含7个外显子、6个内含子,编码的APRT蛋白长212个氨基酸残基,与其他物种来源的APRT序列存在很高的同源性.与大麦、小麦、拟南芥1型及其2型的该蛋白同源性分别为54.9%、54.9%、49.6%和59.5%.经保守结构域搜索发现该蛋白中存在APRT催化结构域.从DNA、mRNA两个水平分析了该基因与水稻温敏核雄性不育(TGMS)的关系,结果表明:受温度诱导,水稻"安农S-1"APRT基因的表达变化可能与温敏核雄性不育表现型具相关性.     

Crystal structures of adenine phosphoribosyltransferase from Leishmania donovani.

The enzyme adenine phosphoribosyltransferase (APRT) functions to salvage adenine by converting it to adenosine-5-monophosphate (AMP). APRT deficiency in humans is a well characterized inborn error of metabolism, and APRT may contribute to the indispensable nutritional role of purine salvage in protozoan parasites, all of which lack de novo purine biosynthesis. We determined crystal structures for APRT from Leishmania donovani in complex with the substrate adenine, the product AMP, and sulfate and citrate ions that appear to mimic the binding of phosphate moieties. Overall, these structures are very similar to each other, although the adenine and AMP complexes show different patterns of hydrogen-bonding to the base, and the active site pocket opens slightly to accommodate the larger AMP ligand. Whereas AMP adopts a single conformation, adenine binds in two mutually exclusive orientations: one orientation providing adenine-specific hydrogen bonds and the other apparently positioning adenine for the enzymatic reaction. The core of APRT is similar to that of other phosphoribosyltransferases, although the adenine-binding domain is quite different. A C-terminal extension, unique to Leishmania APRTs, extends an extensive dimer interface by wrapping around the partner molecule. The active site involves residues from both subunits of the dimer, indicating that dimerization is essential for catalysis.     

APRT from erythrocytes of HGPRT deficient patients: Kinetic,regulatory and thermostability properties

Adenine phosphoribosyltransferase (APRT) has been 1200-fold purified from erythrocytes of a patient with partial hipoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency, Propositus, and in those of a control^HPRT+, with 20% efficiency in both proteins and specific activity of 550 and 243 nmol/h/mgprotein. The specific activity determined in the Propositus enzyme was, in all purification steps, higher than that of the control^HPRT+. Significant changes were found in their thermal stabilities. Half inactivation times at each temperature studied are greater for the Propositus enzyme in the temperature interval 60–80°C. No significant difference has been observed in the affinity constants for adenine and PRPP substrates. Studies on inhibition by the reaction product suggest that AMP is a competitive inhibitor with respect to PRPP in both enzymes, with K_i values of 150 M in Propositus and 220 M in control^HPRT+.     

Internal promoter of the tryptophan operon of Escherichia coli is located in a structural gene

The constitutive low-efficiency promoter site (P₂) near the middle of the tryptophan operon of Escherichia coli has been mapped by analysis of short deletions internal to the trp operon. Comparison of deletions which remove this internal promoter with those which retain it show that P₂ is located within trpD, the region coding for phosphoribosyl anthranilate transferase. P₂ maps near the operator-distal end of trpD, on the operator-proximal side of two trpD point mutants. Comparisons of strains with and without the P₂ site indicate that initiations at this promoter are responsible for synthesis of 80% of the trpC, trpB and trp A polypeptides present in repressed cells.     

Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism

Adenine phosphoribosyltransferase (APRT) is an important enzyme component of the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases rendering this biosynthetic pathway an attractive target for antiparasitic drug design. The recombinant human adenine phosphoribosyltransferase (hAPRT) structure was resolved in the presence of AMP in the active site to 1.76 A resolution and with the substrates PRPP and adenine simultaneously bound to the catalytic site to 1.83 A resolution. An additional structure was solved containing one subunit of the dimer in the apo-form to 2.10 A resolution. Comparisons of these three hAPRT structures with other 'type I' PRTases revealed several important features of this class of enzymes. Our data indicate that the flexible loop structure adopts an open conformation before and after binding of both substrates adenine and PRPP. Comparative analyses presented here provide structural evidence to propose the role of Glu104 as the residue that abstracts the proton of adenine N9 atom before its nucleophilic attack on the PRPP anomeric carbon. This work leads to new insights to the understanding of the APRT catalytic mechanism.     

Adenine salvage activity during callus induction and plant growth

Adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) activity was monitored in crude extracts of Arabidopsis thaliana tissues and callus. Changes in APRT activity during germination were determined within different organs of the mature plant and during callus induction. APRT activity was constitutively expressed in all organs examined. There was an increase in APRT activity detected in seeds beginning 3 days following imbibition, after which the level decreased to that found in leaf tissue of mature plants. There was also an increase in APRT activity early during callus induction. A mutant that lacks APRT activity had a diminished capacity for callus induction in both the presence and absence of exogenous cytokinin. The results are consistent with the hypothesis that an increase in APRT activity is associated with actively dividing cells. The significance of these observations is discussed relative to the role of APRT in adenylate and cytokinin metabolism during plant development.     

Resistance of cultured human fibroblasts and other cells to purine and pyrimidine analogues in relation to mutagenesis detection

In vitro enumeration of diploid human cell variants that are resistant to purine analogues is a possible method of detecting mutagenesis. Their incidences can be increased by the known mutagens, X-rays and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Usefulness of this method depends on the kinds of hereditary changes that confer analogue-resistance on somatic cells. If resistance usually results from changes in genetic material, in vitro studies could be useful indicators of mutagenic effects on somatic cells and germ cells in vivo. If epigenetic changes are primarily responsible for analogue-resistant variants, their enumeration might not provide information relevant to germinal mutations but would still be a useful way to detect induction of general kinds of stable phenotypic changes that could cause cancer. This article outlines hypothetical epigenetic and genetic causes of somatic cell variation and a prospective genetic analysis of human cell variants that are resistant to 8-azaguanine (AG) or 2,6-diaminopurine ( (DAP).Recent evidences and arguments favoring epigenetic origins of resistance to base-analogues are inconclusive. The often cited high rate of changes causing impermeability to BUdR in hamster cells is based on one improperly executed determination. Comparisons of rates of variation conferring BUdR-resistance on cultured haploid and diploid frog cells included diploid variants that did not behave as mutants and ignored major sources of error in estimating mutation rates. AG-resistance could result from recessive mutations in X-chromosomal genes but comparisons of rates of mutation in hamster cells of different ploidies did not provide information about the numbers of X-chromosomes in the variants. Reports that normal rodent HGPRT reappeared in hybrids of enzyme-deficient rodent cells and HGPRT-containing cells of other species or in the rodent cells alone in response to the conditions of cell hybridization did not include adequate controls for reversions in mutant genes of the rodent cells. Questions about the epigenetic and genetic origins of analogue-resistance are mostly unanswered. It remains possible that some kinds of abnormal epigenetic changes cause somatic disease. Specific methods for detecting their occurrence and responsiveness to environmental factors should be devised by focusing efforts on traits that are normally subject to epigenetic regulation. Derepression of genes on the inactive X-chromosome and of liver phenylalanine hydroxylase production are presented as possible examples of abnormal epigenetic changes that could be quantitatively studied by direct selection in vitro.     

APT1, but Not APT2, Codes for a Functional Adenine Phosphoribosyltransferase in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only APT1 could code for a complementing activity. Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that APT1 is the functional gene in S. cerevisiae and that APT2 is a pseudogene.     

Male sterility associated with APRT deficiency in Arabidopsis thaliana results from a mutation in the gene APT1

Four mutants of Arabidopsis thaliana that are deficient in adenine phosphoribosyl transferase (APRT) activity have been isolated by selecting for germination of seeds and growth of the plantlets on a medium containing 2,6-diaminopurine (DAP), a toxic analog of adenine. In all mutants, DAP resistance is due to a recessive nuclear mutation at a locus designated apt. The mutants are male sterile due to pollen abortion after meiosis. Furthermore, it has been shown that metabolism of cytokinins is impaired in the mutant BM3, which has the lowest level of APRT activity among the mutants tested. However, three different cDNAs encoding APRT have been isolated in A. thaliana and this raised the question of the nature of the mutation which results in low APRT activity. The mutation was genetically mapped to chromosome I and lies within 6 cM of the phenotypic marker dis2, indicating that the mutation affects the APT1 gene, a result confirmed by sequencing of mutant alleles. The mutation in the allele apt1-3 is located at the 5′ splicing site of the third intron, and eliminates a BstNI restriction site, as verified by Southern blotting and PCR fragment length analysis.     

Mutation at the APRT locus in Friend erythroleukaemia cells 2. Sensitivity to mitomycin C induced cytogenetic damage

Clone 707 of the Friend cell was compared with an APRT-deficient subclone for sensitivity to cell killing and the induction of cytogenetic aberrations by mitomycin C (MMC). Two 16-h doses of MMC were used, 0.1 and 0.15 μg/ml and cells were scored for aberrations at 16, 33 and 44 h post-treatment. The APRT-deficient subclone showed increased cell killing, a higher frequency of aberrations and a higher frequency of cells with severe cytogenetic damage. It is proposed that APRT may play a role in balancing deoxyribonucleoside triphosphate pools for DNA-repair processes.     

Adenine phosphoribosyltransferase: A simple spectrophotometric assay and the incidence of mutation in the normal population

The significance of partial deficiency of erythrocyte adenine phosphoribosyltransferase (APRT), reported in a number of subjects with gout, has been investigated by studying its incidence in 700 normal blood donors. Three clearly deficient subjects were found, an incidence not significantly different from that in patients with abnormalities of urate metabolism. A new assay method for APRT is described in which an erythrocyte lysate is incubated with adenine and phosphoribosylpyrophosphate (PRPP) for a given time; both hemoglobin and adenine nucleotide (AMP) are then precipitated with lanthanum phosphate; the change in absorbance of adenine at 260 nm reflects the extent of its conversion to AMP by APRT.This work was supported by the National Health and Medical Research Council of Australia.     

Identification of Two Novel Mutations in Adenine Phosphoribosyltransferase Gene in Patients with 2,8‐Dihydroxyadenine Urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani

Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation of adenine from alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to form AMP and PP(i). Three-dimensional structures of the dimeric APRT enzyme from Leishmania donovani (LdAPRT) bear many similarities to other members of the type 1 phosphoribosyltransferase family but do not reveal the structural basis for catalysis (Phillips, C. L., Ullman, B., Brennan, R. G., and Hill, C. P. (1999) EMBO J. 18, 3533-3545). To address this issue, a steady state and transient kinetic analysis of the enzyme was performed in order to determine the catalytic mechanism. Initial velocity and product inhibition studies indicated that LdAPRT follows an ordered sequential mechanism in which PRPP is the first substrate to bind and AMP is the last product to leave. This mechanistic model was substantiated by equilibrium isotope exchange and fluorescence binding studies, which provided dissociation constants for the LdAPRT-PRPP and LdAPRT-AMP binary complexes. Pre-steady-state kinetic analysis of the forward reaction revealed a burst in product formation indicating that phosphoribosyl transfer proceeds rapidly relative to some rate-limiting product release event. Transient fluorescence competition experiments enabled measurement of rates of binary complex dissociation that implicated AMP release as rate-limiting for the forward reaction. Kinetics of product ternary complex formation were evaluated using the fluorophore formycin AMP and established rate constants for pyrophosphate binding to the LdAPRT-formycin AMP complex. Taken together, these data enabled the complete formulation of an ordered bi-bi kinetic mechanism for LdAPRT in which all of the rate constants were either measured or calculated.