Adenylosuccinate lyase catalyzes 2 distinct but chemically related steps in purine biosynthesis: the conversion of adenylosuccinate to AMP + fumarate, and the cleavage of 5-aminoimidazole-4-(N-succinylocarboxamide)ribonucleotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + fumarate. The importance of the metabolic role played by adenylosuccinate lyase (ASL) is indicated by the severity of the symptoms of ASL deficiency in humans; the disease is associated with mental retardation, psychomotor delay, epilepsy and autism. ASL is isolated as a tetramer of identical subunits. Crystals of enzyme in the absence of substrate or inhibitor have been prepared from 3 bacterial species and high-resolution structures have been reported for two of them. In Dr. Colman's laboratory, affinity labeling and site-directed mutagenesis of the Bacillus subtilis ASL led to the identification of 3 His (1168, H89 and H141) as critical for catalysis, 2 of which likely function as general acid/general base during the reaction and are contributed by 2 different subunits. However, examination of the structure of adenylosuccinate lyase leads to the hypothesis that in an enzyme tetramer, 3 subunits provide amino acids to each of the 4 active sites. The overall goal of this project is to understand the major chemical and structural contributions to catalysis by normal ASL and the molecular basis for decreased activity in patients with ASL deficiency. Following questions are being now asked: which additional amino acids of ASL participate in catalysis and/or substrate binding, and which subunits provide these residues? These issues will be approached by site-directed mutagenesis of amino acids of B. subtilis ASL postulated to be at the active site. Targets of mutagenesis will be selected on the basis of conservation among the sequences of ASL from 28 species and proximity in the crystal structures to the active site. Mutant enzymes will be expressed, purified to homogeneity and extensively characterized by kinetics, binding and biophysical measurements. Complementation experiments will be conducted in which pairs of different, inactive mutants are mixed and tested for restoration of activity in hybrid tetramers. Bifunctional affinity labels will be evaluated to ascertain whether crosslinking of subunits occurs. To elucidate the molecular basis of ASL deficiency, mutants of B. subtilis ASL will be constructed with amino acid substitutions equivalent to those in human patients with ASL deficiency. The mutant enzymes will be purified and extensively characterized. The intention is to use these mutant enzymes as models to evaluate the structural/chemical basis of a human genetic defect.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Biochemistry Study Section (BIO)
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Sechi, Salvatore
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University of Delaware
Schools of Arts and Sciences
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De Zoysa Ariyananda, Lushanti; Antonopoulos, Christina; Currier, Jenna et al. (2011) In vitro hybridization and separation of hybrids of human adenylosuccinate lyase from wild-type and disease-associated mutant enzymes. Biochemistry 50:1336-46
Ariyananda, Lushanti De Zoysa; Lee, Peychii; Antonopoulos, Christina et al. (2009) Biochemical and biophysical analysis of five disease-associated human adenylosuccinate lyase mutants. Biochemistry 48:5291-302
Sivendran, Sharmila; Colman, Roberta F (2008) 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. Protein Sci 17:1162-74
De Zoysa Ariyananda, Lushanti; Colman, Roberta F (2008) Evaluation of types of interactions in subunit association in Bacillus subtilis adenylosuccinate lyase. Biochemistry 47:2923-34
Sivendran, Sharmila; Segall, Mark L; Rancy, Pumtiwitt C et al. (2007) Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase. Protein Sci 16:1700-7
Segall, Mark L; Cashman, Meghan A; Colman, Roberta F (2007) Important roles of hydroxylic amino acid residues in the function of Bacillus subtilis adenylosuccinate lyase. Protein Sci 16:441-8
Spiegel, Erin K; Colman, Roberta F; Patterson, David (2006) Adenylosuccinate lyase deficiency. Mol Genet Metab 89:19-31
Sivendran, Sharmila; Patterson, David; Spiegel, Erin et al. (2004) Two novel mutant human adenylosuccinate lyases (ASLs) associated with autism and characterization of the equivalent mutant Bacillus subtilis ASL. J Biol Chem 279:53789-97
Segall, Mark L; Colman, Roberta F (2004) Gln212, Asn270, and Arg301 are critical for catalysis by adenylosuccinate lyase from Bacillus subtilis. Biochemistry 43:7391-402
Palenchar, Jennifer Brosius; Colman, Roberta F (2003) Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role. Biochemistry 42:1831-41

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