Publications

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

  Tsai M., Koo J., Yip P., Colman R.F., Segall M.L., Howell P.L.

  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 pathwa ... >> More

  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. << Less

  J. Mol. Biol. 370:541-554(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Ray S.P., Deaton M.K., Capodagli G.C., Calkins L.A., Sawle L., Ghosh K., Patterson D., Pegan S.D.

  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-aminoimi ... >> More

  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. << Less

  Biochemistry 51:6701-6713(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• 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.

  Sivendran S., Colman R.F.

  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 ... >> More

  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. << Less

  Protein Sci 17:1162-1174(2008) [PubMed] [EuropePMC]

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

  Segall M.L., Colman R.F.

  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-t ... >> More

  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. << Less

  Biochemistry 43:7391-7402(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expression, purification, and characterization of stable, recombinant human adenylosuccinate lyase.

  Lee P., Colman R.F.

  The full length human adenylosuccinate lyase gene was generated by a PCR method using a plasmid encoding a truncated human enzyme as template, and was cloned into a pET-14b vector. Human adenylosuccinate lyase was overexpressed in Escherichia coli Rosetta 2(DE3)pLysS as an N-terminal histidine-tag ... >> More

  The full length human adenylosuccinate lyase gene was generated by a PCR method using a plasmid encoding a truncated human enzyme as template, and was cloned into a pET-14b vector. Human adenylosuccinate lyase was overexpressed in Escherichia coli Rosetta 2(DE3)pLysS as an N-terminal histidine-tagged protein and was purified to homogeneity by a nickel-nitriloacetic acid column at room temperature. The histidine tag was removed from the human enzyme by thrombin digestion and the adenylosuccinate lyase was purified by Sephadex G-100 gel filtration. The histidine-tagged and non-tagged adenylosuccinate lyases exhibit similar values of Vmax and Km for S-AMP. Analytical ultracentrifugation and circular dichroism revealed, respectively, that the histidine-tagged enzyme is in tetrameric form with a molecular weight of 220 kDa and contains predominantly alpha-helical structure. This is the first purification procedure to yield a stable form of human adenylosuccinate lyase. The enzyme is stable for at least 5 days at 25 degrees C, and upon rapid freezing and thawing. Temperature as well as reducing agent (DTT) play critical roles in determining the stability of the human adenylosuccinate lyase. << Less

  Protein Expr. Purif. 51:227-234(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Adenylosuccinate lyase from Artemia embryos. Purification and properties.

  Pinto R.M., Faraldo A., Fernandez A., Canales J., Sillero A., Sillero M.A.

  In crude extracts, the molecular form of adenylosuccinate lyase is pH-dependent as studied by gel filtration and sucrose gradient centrifugation. At pH values of 8.7 and 6.5, the enzyme exhibits molecular forms of 200 kDa and larger than 500 kDa, respectively. At pH values of 7.0-7.5, forms of int ... >> More

  In crude extracts, the molecular form of adenylosuccinate lyase is pH-dependent as studied by gel filtration and sucrose gradient centrifugation. At pH values of 8.7 and 6.5, the enzyme exhibits molecular forms of 200 kDa and larger than 500 kDa, respectively. At pH values of 7.0-7.5, forms of intermediate molecular weight were detected. Interconversion among the different molecular forms of adenylosuccinate lyase was not achieved when the enzyme was subjected to two successive chromatographic steps on Sepharose CL-6B using elution buffers at two different pH values. A unique form of 200 kDa was observed, regardless of the pH of the buffer used, upon either gel filtration or sucrose gradient centrifugation in the presence of 1 M NaCl. The enzyme from Artemia cysts was purified to homogeneity. It had molecular mass of 200 kDa and gave a single band of 56 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Km values for adenylosuccinate, AMP, and fumarate were 1, 36, and 350 microM, respectively. The enzyme exhibits a Uni Bi-ordered mechanism and is maximally active at a pH value of 8.0-8.5. For maximum activity, the enzyme requires an ionic strength equivalent to 80 mM KCI. The isoelectric point determined by chromatofocusing was 5.04. << Less

  J Biol Chem 258:12513-12519(1983) [PubMed] [EuropePMC]

• Escherichia coli purB gene: cloning, nucleotide sequence, and regulation by purR.

  He B., Smith J.M., Zalkin H.

  Escherichia coli purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of IMP and also the final reaction in the two-step sequence from IMP to AMP. Gene purB was cloned and found to encode an ASL protein of 435 amino acids having a calcula ... >> More

  Escherichia coli purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of IMP and also the final reaction in the two-step sequence from IMP to AMP. Gene purB was cloned and found to encode an ASL protein of 435 amino acids having a calculated molecular weight of 49,225. E. coli ASL is homologous to the corresponding enzymes from Bacillus subtilis and chickens and also to fumarase from B. subtilis. Gene phoP is 232 bp downstream of purB. Gene purB is regulated threefold by the purine pool and purR. Transcriptional regulation of purB involves binding of the purine repressor to the 16-bp conserved pur regulon operator. The purB operator is 224 bp downstream of the transcription start site and overlaps codons 62 to 67 in the protein-coding sequence. << Less

  J. Bacteriol. 174:130-136(1992) [PubMed] [EuropePMC]

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

  Bulusu V., Srinivasan B., Bopanna M.P., Balaram H.

  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 (AIC ... >> More

  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. << Less

  Biochim Biophys Acta 1794:642-654(2009) [PubMed] [EuropePMC]