Arg40 is critical for stability and activity of Adenylosuccinate lyase; a purine salvage enzyme from Leishmania donovani.

Purine salvage enzymes have been of significant interest in anti-Leishmanial drug development due to the parasite's critical dependence on this pathway for the supply of nucleotides in the absence of a de novo purine synthesis pathway. Adenylosuccinate lyase (ADSL) one of the key enzymes in this pathway is a homo-tetramer, where the active site is formed by residues from three distinct subunits. Analysis of the subunit interfaces of LdADSL, revealed a conserved Arg40 forming critical inter-subunit interactions and also involved in substrate binding. We hypothesized that mutating this residue can affect both the structural stability and activity of the enzyme. In our study, we used biochemical, biophysical, and computational simulation approaches to understand the structural and functional role of Arg40 in LdADSL. We have replaced Arg40 with an Ala and Glu using site directed mutagenesis. The mutant enzymes were similar to wild-type enzyme in secondary structure and subunit association. Thermal shift assays indicated that the mutations affected the protein stability. Both mutants showed decreased specific activities in both forward and reverse directions with significantly weakened affinities towards succinyl-adenosine monophosphate (SAMP). The mutations resulted in changes in C3 loop conformation and D3 domain rotation. Consequently, the orientation of the active site amino acid residues changed resulting in compromised activity and stability. Studies so far have majorly focused on the ADSL active site for designing drugs against it. Our work indicates that an alternative inhibitory mechanism for the enzyme can be designed by targeting the inter-subunit interface.

MicroRNA-21 guide and passenger strand regulation of adenylosuccinate lyase-mediated purine metabolism promotes transition to an EGFR-TKI-tolerant persister state.

In EGFR-mutant lung cancer, drug-tolerant persister cells (DTPCs) show prolonged survival when receiving EGFR tyrosine kinase inhibitor (TKI) treatments. They are a likely source of drug resistance, but little is known about how these cells tolerate drugs. Ribonucleic acids (RNAs) molecules control cell growth and stress responses. Nucleic acid metabolism provides metabolites, such as purines, supporting RNA synthesis and downstream functions. Recently, noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), have received attention due to their capacity to repress gene expression via inhibitory binding to downstream messenger RNAs (mRNAs). Here, our study links miRNA expression to purine metabolism and drug tolerance. MiR-21-5p (guide strand) is a commonly upregulated miRNA in disease states, including cancer and drug resistance. However, the expression and function of miR-21-3p (passenger strand) are not well understood. We found that upregulation of miR-21-5p and miR-21-3p tune purine metabolism leading to increased drug tolerance. Metabolomics data demonstrated that purine metabolism was the top pathway in the DTPCs compared with the parental cells. The changes in purine metabolites in the DTPCs were partially rescued by targeting miR-21. Analysis of protein levels in the DTPCs showed that reduced expression of adenylosuccinate lyase (ADSL) was reversed after the miR-21 knockdown. ADSL is an essential enzyme in the de novo purine biosynthesis pathway by converting succino-5-aminoimidazole-4-carboxamide riboside (succino-AICAR or SAICAR) to AICAR (or acadesine) as well as adenylosuccinate to adenosine monophosphate (AMP). In the DTPCs, miR-21-5p and miR-21-3p repress ADSL expression. The levels of top decreased metabolite in the DTPCs, AICAR was reversed when miR-21 was blocked. AICAR induced oxidative stress, evidenced by increased reactive oxygen species (ROS) and reduced expression of nuclear factor erythroid-2-related factor 2 (NRF2). Concurrently, miR-21 knockdown induced ROS generation. Therapeutically, a combination of AICAR and osimertinib increased ROS levels and decreased osimertinib-induced NRF2 expression. In a MIR21 knockout mouse model, MIR21 loss-of-function led to increased purine metabolites but reduced ROS scavenging capacity in lung tissues in physiological conditions. Our data has established a link between ncRNAs, purine metabolism, and the redox imbalance pathway. This discovery will increase knowledge of the complexity of the regulatory RNA network and potentially enable novel therapeutic options for drug-resistant patients.

Disorders of purine biosynthesis metabolism.

Purines are essential molecules that are components of vital biomolecules, such as nucleic acids, coenzymes, signaling molecules, as well as energy transfer molecules. The de novo biosynthesis pathway starts from phosphoribosylpyrophosphate (PRPP) and eventually leads to the synthesis of inosine monophosphate (IMP) by means of 10 sequential steps catalyzed by six different enzymes, three of which are bi-or tri-functional in nature. IMP is then converted into guanosine monophosphate (GMP) or adenosine monophosphate (AMP), which are further phosphorylated into nucleoside di- or tri-phosphates, such as GDP, GTP, ADP and ATP. This review provides an overview of inborn errors of metabolism pertaining to purine synthesis in humans, including either phosphoribosylpyrophosphate synthetase (PRS) overactivity or deficiency, as well as adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), and adenylosuccinate synthetase (ADSS) deficiencies. ITPase deficiency is being described as well. The clinical spectrum of these disorders is broad, including neurological impairment, such as psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscle presentations or consequences of hyperuricemia, such as gouty arthritis or kidney stones. Clinical signs are often nonspecific and, thus, overlooked. It is to be hoped that this is likely to be gradually overcome by using sensitive biochemical investigations and next-generation sequencing technologies.

Targeting the De Novo Purine Synthesis Pathway Through Adenylosuccinate Lyase Depletion Impairs Liver Cancer Growth by Perturbing Mitochondrial Function.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is among the most common cancer types worldwide, yet patients with HCC have limited treatment options. There is an urgent need to identify drug targets that specifically inhibit the growth of HCC cells. APPROACH AND RESULTS: We used a CRISPR library targeting ~2,000 druggable genes to perform a high-throughput screen and identified adenylosuccinate lyase (ADSL), a key enzyme involved in the de novo purine synthesis pathway, as a potential drug target for HCC. ADSL has been implicated as a potential oncogenic driver in some cancers, but its role in liver cancer progression remains unknown. CRISPR-mediated knockout of ADSL impaired colony formation of liver cancer cells by affecting AMP production. In the absence of ADSL, the growth of liver tumors is retarded in vivo. Mechanistically, we found that ADSL knockout caused S-phase cell cycle arrest not by inducing DNA damage but by impairing mitochondrial function. Using data from patients with HCC, we also revealed that high ADSL expression occurs during tumorigenesis and is linked to poor survival rate. CONCLUSIONS: Our findings uncover the role of ADSL-mediated de novo purine synthesis in fueling mitochondrial ATP production to promote liver cancer cell growth. Targeting ADSL may be a therapeutic approach for patients with HCC.

Prolyl hydroxylase substrate adenylosuccinate lyase is an oncogenic driver in triple negative breast cancer.

Protein hydroxylation affects protein stability, activity, and interactome, therefore contributing to various diseases including cancers. However, the transiency of the hydroxylation reaction hinders the identification of hydroxylase substrates. By developing an enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify adenylosuccinate lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is higher in TNBC than other breast cancer subtypes or normal breast tissues. ADSL knockout impairs TNBC cell proliferation and invasiveness in vitro and in vivo. An integrated transcriptomics and metabolomics analysis reveals that ADSL activates the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL proline 24 hydroxylation. Hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating cMYC protein level. Our findings highlight the role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.

Mapping Post-Translational Modifications of de Novo Purine Biosynthetic Enzymes: Implications for Pathway Regulation.

Purines represent a class of essential metabolites produced by the cell to maintain cellular homeostasis and facilitate cell proliferation. In times of high purine demand, the de novo purine biosynthetic pathway is activated; however, the mechanisms that facilitate this process are largely unknown. One plausible mechanism is through intracellular signaling, which results in enzymes within the pathway becoming post-translationally modified to enhance their individual enzyme activities and the overall pathway metabolic flux. Here, we employ a proteomic strategy to investigate the extent to which de novo purine biosynthetic pathway enzymes are post-translationally modified in 293T cells. We identified 7 post-translational modifications on 135 residues across the 6 human pathway enzymes. We further asked whether there were differences in the post-translational modification state of each pathway enzyme isolated from cells cultured in the presence or absence of purines. Of the 174 assigned modifications, 67% of them were only detected in one experimental growth condition in which a significant number of serine and threonine phosphorylations were noted. A survey of the most-probable kinases responsible for these phosphorylation events uncovered a likely AKT phosphorylation site at residue Thr397 of PPAT, which was only detected in cells under purine-supplemented growth conditions. These data suggest that this modification might alter enzyme activity or modulate its interaction(s) with downstream pathway enzymes. Together, these findings propose a role for post-translational modifications in pathway regulation and activation to meet intracellular purine demand.

Adenylosuccinate lyase enhances aggressiveness of endometrial cancer by increasing killer cell lectin-like receptor C3 expression by fumarate.

Adenylosuccinate lyase (ADSL) is an enzyme that plays important roles in de novo purine synthesis. Although ADSL was reported to be upregulated in various malignancies, such as colorectal, breast, and prostate cancer, as well as gliomas, the mechanism by which elevated ADSL expression contributes to cancer has not been elucidated. We previously performed a shotgun proteomics analysis to characterize specific proteins associated with the properties of the aldehyde dehydrogenase (ALDH)-high cell population, which was reported to be involved in tumorigenic potential, and showed that ADSL expression is upregulated in the ALDH-high population of endometrial cancer. Here, we showed that ADSL is involved in endometrial cancer aggressiveness by regulating expression of killer cell lectin-like receptor C3 (KLRC3), which is a receptor expressed on natural killer cells. Immunohistochemical analysis indicated that ADSL expression increased as endometrioid carcinoma specimens became more poorly differentiated and higher degree of primary tumor progression. Knockdown of ADSL in endometrial cancer cells decreased cell proliferation, migration, and invasive capability, and caused the cells to adopt a more rounded shape. DNA microarray analysis and quantitative real-time PCR showed that KLRC3 expression was decreased in ADSL knockdown cells. Knockdown of KLRC3 in endometrial cancer cells resulted in the same phenotype as knockdown of ADSL. Moreover, fumarate, which could be produced by ADSL and was recently shown to be an oncometabolite, recovered KLRC3 expression in ADSL knockdown cells, suggesting that fumarate produced by ADSL could regulate KLRC3 expression. Our findings indicate that ADSL enhances cell proliferation, migration, and invasive capability through regulation of KLRC3 expression by fumarate.

Structural and kinetic analysis of Schistosoma mansoni Adenylosuccinate Lyase (SmADSL).

Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5'-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites are formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway.

Adenylosuccinate Is an Insulin Secretagogue Derived from Glucose-Induced Purine Metabolism.

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet ß cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human ß cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in ß cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing ß cell dysfunction in T2D.

Inherent properties of adenylosuccinate lyase could explain S-Ado/SAICAr ratio due to homozygous R426H and R303C mutations.

Adenylosuccinate lyase (ADSL) is a homotetrameric enzyme involved in the de novo purine biosynthesis pathway and purine nucleotide cycle. Missense mutations in the protein lead to ADSL deficiency, an inborn error of purine metabolism characterized by neurological and physiological symptoms. ADSL deficiency is biochemically diagnosed by elevated levels of succinylaminoimidazolecarboxamide riboside (SAICAr) and succinyladenosine (S-Ado), the dephosphorylated derivatives of the substrates. S-Ado/SAICAr ratios have been associated with three phenotypic groups. Different hypotheses to explain these ratios have been proposed. Recent studies have focused on measuring activity on the substrates independently. However, it is important to examine mixtures of the substrates to determine if mutations affect enzyme activity on both substrates similarly in these conditions. The two substrates may experience an indirect communication due to being acted upon by the same enzyme, altering their activities from the non-competitive case. In this study, we investigate this hidden coupling between the two substrates. We chose two mutations that represent extremes of the phenotype, R426H and R303C. We describe a novel electrochemical-detection method of measuring the kinetic activity of ADSL in solution with its two substrates at varying concentration ratios. Furthermore, we develop an enzyme kinetic model to predict substrate activity from a given ratio of substrate concentrations. Our findings indicate a non-linear dependence of the activities on the substrate ratios due to competitive binding, distinct differences in the behaviors of the different mutations, and S-Ado/SAICAr ratios in patients could be explained by inherent properties of the mutant enzyme.

Metabolic disorders of purine metabolism affecting the nervous system.

The purines are a group of molecules used by all cells for many vital biochemical processes including energy-requiring enzymatic reactions, cofactor-requiring reactions, synthesis of DNA or RNA, signaling pathways within and between cells, and other processes. Defects in some of the enzymes of purine metabolism are known to be associated with specific clinical disorders, and neurological problems may be a presenting sign or the predominant clinical problem for several of them. This chapter describes three disorders for which the clinical features and metabolic basis are well characterized. Deficiency of adenylosuccinate-lyase (ADSL) causes psychomotor retardation, epilepsy, and autistic features. Lesch-Nyhan disease is caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and is characterized by hyperuricemia, motor and cognitive disability, and self-injurious behavior. Deficiency of myoadenylate deaminase (mAMPD) is associated with myopathic features. In addition to these disorders, several other disorders are briefly summarized. These include defects of phosphoribosylpyrophosphate synthase, adenosine deaminase (ADA), purine nucleoside phosphorylase (PND), deoxyguanosine kinase (dGK), or IMP dehydrogenase (IMPDH). Each of these disorders provides an unusual window on the unique importance of purine metabolism for function of different parts of the nervous system.

Structural and biochemical characterization of human adenylosuccinate lyase (ADSL) and the R303C ADSL deficiency-associated mutation.

Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive disorder, which causes a defect in purine metabolism resulting in neurological and physiological symptoms. ADSL executes two nonsequential steps in the de novo synthesis of AMP: the conversion of phosphoribosylsuccinyl-aminoimidazole carboxamide (SAICAR) to phosphoribosylaminoimidazole carboxamide, which occurs in the de novo synthesis of IMP, and the conversion of adenylosuccinate to AMP, which occurs in the de novo synthesis of AMP and also in the purine nucleotide cycle, using the same active site. Mutation of ADSL's arginine 303 to a cysteine is known to lead to ADSL deficiency. Interestingly, unlike other mutations leading to ADSL deficiency, the R303C mutation has been suggested to more significantly affect the enzyme's ability to catalyze the conversion of succinyladenosine monophosphate than that of SAICAR to their respective products. To better understand the causation of disease due to the R303C mutation, as well as to gain insights into why the R303C mutation potentially has a disproportional decrease in activity toward its substrates, the wild type (WT) and the R303C mutant of ADSL were investigated enzymatically and thermodynamically. Additionally, the X-ray structures of ADSL in its apo form as well as with the R303C mutation were elucidated, providing insight into ADSL's cooperativity. By utilizing this information, a model for the interaction between ADSL and SAICAR is proposed.

Neurological disorders of purine and pyrimidine metabolism.

Purines and pyrimidines, regarded for a long time only as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, gained increasing attention since genetically determined aberrations in their metabolism were associated clinically with various degrees of mental retardation and/or unexpected and often devastating neurological dysfunction. In most instances the molecular mechanisms underlying neurological symptoms remain undefined. This suggests that nucleotides and nucleosides play fundamental but still unknown roles in the development and function of several organs, in particular central nervous system. Alterations of purine and pyrimidine metabolism affecting brain function are spread along both synthesis (PRPS, ADSL, ATIC, HPRT, UMPS, dGK, TK), and breakdown pathways (5NT, ADA, PNP, GCH, DPD, DHPA, TP, UP), sometimes also involving pyridine metabolism. Explanations for the pathogenesis of disorders may include both cellular and mitochondrial damage: e.g. deficiency of the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase are associated to the most severe pathologies, the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to impairment of mitochondrial functions. This review gathers the presently known inborn errors of purine and pyrimidine metabolism that manifest neurological syndromes, reporting and commenting on the available hypothesis on the possible link between specific enzymatic alterations and brain damage. Such connection is often not obvious, and though investigated for many years, the molecular basis of most dysfunctions of central nervous system associated to purine and pyrimidine metabolism disorders are still unexplained.

Purine Salvage Pathway in Mycobacterium tuberculosis.

Millions of deaths worldwide are caused by the aetiological agent of tuberculosis, Mycobacterium tuberculosis. The increasing prevalence of this disease, the emergence of drug-resistant strains, and the devastating effect of human immunodeficiency virus coinfection have led to an urgent need for the development of new and more efficient antimycobacterial drugs. The modern approach to the development of new chemical compounds against complex diseases, especially the neglected endemic ones, such as tuberculosis, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a specific target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, and (iii) the development of compounds with selective toxicity. The present review describes the enzymes of the purine salvage pathway in M. tuberculosis as attractive targets for the development of new antimycobacterial agents. Enzyme kinetics and structural data have been included to provide a thorough knowledge on which to base the search for compounds with biological activity. We have focused on the mycobacterial homologues of this pathway as potential targets for the development of new antitubercular agents.

Elucidation of the substrate specificity, kinetic and catalytic mechanism of adenylosuccinate lyase from Plasmodium falciparum.

Adenylosuccinate lyase (ASL) catalyzes two distinct but chemically similar reactions in purine biosynthesis. The first, exclusive to the de novo pathway involves the cleavage of 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and fumarate and the second common to both de novo and the salvage pathways involves the cleavage of succinyl-adenosine monophosphate (SAMP) to AMP and fumarate. A detailed kinetic and catalytic mechanism of the recombinant His-tagged ASL from Plasmodium falciparum (PfASL) is presented here. Initial velocity kinetics, product inhibition studies and transient kinetics indicate a Uni-Bi rapid equilibrium ordered mechanism. Substrate and solvent isotope effect studies implicate the process of C(gamma)-N bond cleavage to be rate limiting. Interestingly, the effect of pH on k(cat) and k(cat)/K(m) highlight ionization of the base only in the enzyme substrate complex and not in the enzyme alone, thereby implicating the pivotal role of the substrate in the activation of the catalytic base. Site-directed mutagenesis implicates a key role for the conserved serine (S298) in catalysis. Despite the absence of a de novo pathway for purine synthesis and most importantly, the absence of other enzymes that can metabolise AICAR in P. falciparum, PfASL catalyzes the SAICAR cleavage reaction with kinetic parameters similar to those of SAMP reaction and binds AICAR with affinity similar to that of AMP. The presence of this catalytic feature allows the use of AICAR or its analogues as inhibitors of PfASL and hence, as novel putative anti-parasitic agents. In support of this, we do see a dose dependent inhibition of parasite growth in the presence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAriboside) with half-maximal inhibition at 167+/-5 microM.

Effect of a new non-cleavable substrate analog on wild-type and serine mutants in the signature sequence of adenylosuccinate lyase of Bacillus subtilis and Homo sapiens.

Adenylosuccinate lyase (ASL) catalyzes two beta-elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2'(3'), 5'-diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (K(I) approximately 0.1 microM), indicating that the two substrates occupy the same active site. Binding studies show that both the B. subtilis and human ASLs bind up to 4 mol of APBADP per mole of enzyme tetramer and that both enzymes exhibit cooperativity: negative for B. subtilis ASL and positive for human ASL. Mutant B. subtilis ASLs, with replacements for residues previously identified as critical for catalysis, bind the substrate analog similarly to wild-type ASL. Two serines in a flexible loop of ASL have been proposed to play roles in catalysis because they are close to the substrate in the crystal structure of Escherichia coli ASL. We have now mutated the corresponding serines to alanines in B. subtilis and human ASL to evaluate their involvement in enzyme function. Kinetic data reveal that human Ser(289) and B. subtilis Ser(262) and Ser(263) are essential for catalysis, while the ability of these Ser mutants to bind APBADP suggests that they do not contribute to substrate affinity. Although these serines are not visible in the crystal structure of human adenylosuccinate lyase complexed with substrate or products (PDB #2VD6), they may be interacting with the active sites.

Substrate and product complexes of Escherichia coli adenylosuccinate lyase provide new insights into the enzymatic mechanism.

Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimidazole- (N-succinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly alpha-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall beta-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP.FUM) complexes have been determined to 2.0, 1.85, and 2.0 A resolution, respectively. The H171A-ADS and H171N-AMP.FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287-303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observed in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.

Mapping of the bovine genes of the de novo AMP synthesis pathway.

Summary The purine nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) are critical for energy metabolism, cell signalling and cell reproduction. Despite their essential function, little is known about the regulation and in vivo expression pattern of the genes involved in the de novo purine synthesis pathway. The complete coding region of the bovine phosphoribosylaminoimidazole carboxylase gene (PAICS), which catalyses steps 6 and 7 of the de novo purine biosynthesis pathway, as well as bovine genomic sequences of the six other genes in the pathway producing inosine monophosphate (IMP) and AMP [phosphoribosyl pyrophosphate amidotransferase (PPAT), phosphoribosylglycinamide formyltransferase (GART), phosphoribosylformylglycinamidine synthase (PFAS), adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) and adenylosuccinate synthase (ADSS)], were identified. The genes were mapped to segments of six different bovine chromosomes using a radiation hybrid (RH) cell panel. The gene PPAT, coding for the presumed rate-limiting enzyme of the purine de novo pathway was closely linked to PAICS on BTA6. These, and the other bovine locations i.e. GART at BTA1, PFAS at BTA19, ADSL at BTA5, ATIC at BTA2 and ADSS at BTA16, are in agreement with published comparative maps of cattle and man. PAICS and PPAT genes are known to be closely linked in human, rat and chicken. Previously, an expressed sequence fragment of PAICS (Bos taurus corpus luteum, BTCL9) was mapped to BTA13. By isolation and characterization of a BAC clone, we have now identified a PAICS processed pseudogene sequence (psiPAICS) on BTA13. Processed pseudogene sequences of PAICS and other genes of the purine biosynthesis pathway were identified in several mammalian species, indicating that the genes of this pathway have been susceptible to retrotransposition. The seven bovine genes are expressed at a higher level in testicular and ovary tissues compared with skeletal muscle.

Gln212, Asn270, and Arg301 are critical for catalysis by adenylosuccinate lyase from Bacillus subtilis.

In adenylosuccinate lyase from Bacillus subtilis, Gln(212), Asn(270), and Arg(301) are conserved and located close to the succinyl moiety of docked adenylosuccinate. We constructed mutant enzymes with Gln(212) replaced by Glu and Met, Asn(270) by Asp and Leu, and Arg(301) by Gln or Lys. The wild-type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity. The specific activities of the Q212M and the 270 and 301 mutant enzymes were decreased more than 3000-fold as compared to the wild type. Only Q212E retained sufficient activity for determination of its kinetic parameters: V(max) was decreased approximately 1000-fold, and K(m) was increased 6-fold, as compared to the wild-type enzyme. Adenylosuccinate binding studies of the other mutants revealed greatly weakened affinities that contributed to, but did not account entirely for, the loss of activity. These mutant enzymes did not differ greatly from the wild-type enzyme in secondary structure or subunit association state, as shown by circular dichroism spectroscopy and light-scattering photometry. Incubation of pairs of inactive mutant enzymes led to reconstitution of some functional sites by subunit complementation, with recovery of up to 25% of the specific activity of the wild-type enzyme. Subunit complementation occurs only if the two mutations are contributed to the active site by different subunits. Thus, mixing Q212E with N270L enzyme yielded a specific activity of approximately 20% of the wild-type enzyme, while mixing Q212M with R301K enzyme did not restore activity. As supported by computer modeling, the studies presented here indicate that Gln(212), Asn(270), and Arg(301) are indispensable to catalysis by adenylosuccinate lyase and probably interact noncovalently with the carboxylate anions of the substrates 5-aminoimidazole-4(N-succinylocarboxamide)ribonucleotide and adenylosuccinate, optimizing their bound orientations.

Mutation of a nuclear respiratory factor 2 binding site in the 5' untranslated region of the ADSL gene in three patients with adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL; also called "adenylosuccinase") catalyzes two steps in the synthesis of purine nucleotides: (1) the conversion of succinylaminoimidazolecarboxamide ribotide into aminoimidazolecarboxamide ribotide and (2) the conversion of adenylosuccinate into adenosine monophosphate. ADSL deficiency, a recessively inherited disorder, causes variable-but most often severe-mental retardation, frequently accompanied by epilepsy and/or autism. It is characterized by the accumulation, in body fluids, of succinylaminoimidazolecarboxamide riboside and succinyladenosine, the dephosphorylated derivatives of the two substrates of the enzyme. Analysis of the ADSL gene of three unrelated patients with ADSL deficiency, in whom one of the ADSL alleles displayed a normal coding sequence, revealed a -49T-->C mutation in the 5' untranslated region of this allele. Measurements of the amount of mRNA transcribed from the latter allele showed that it was reduced to approximately 33% of that transcribed from the alleles mutated in their coding sequence. Further investigations showed that the -49T-->C mutation provokes a reduction to 25% of wild-type control of promoter function, as evaluated by luciferase activity and mRNA level in transfection experiments. The mutation also affects the binding of nuclear respiratory factor 2 (NRF-2), a known activator of transcription, as assessed by gel-shift studies. Our findings indicate that a mutation of a regulatory region of the ADSL gene might be an unusually frequent cause of ADSL deficiency, and they suggest a role for NRF-2 in the gene regulation of the purine biosynthetic pathway.