Publications

• Crystal structure and allosteric regulation of the cytoplasmic Escherichia coli L-asparaginase I.

  Yun M.K., Nourse A., White S.W., Rock C.O., Heath R.J.

  AnsA is the cytoplasmic asparaginase from Escherichia coli involved in intracellular asparagine utilization. Analytical ultracentifugation and X-ray crystallography reveal that AnsA forms a tetrameric structure as a dimer of two intimate dimers. Kinetic analysis of the enzyme reveals that AnsA is ... >> More

  AnsA is the cytoplasmic asparaginase from Escherichia coli involved in intracellular asparagine utilization. Analytical ultracentifugation and X-ray crystallography reveal that AnsA forms a tetrameric structure as a dimer of two intimate dimers. Kinetic analysis of the enzyme reveals that AnsA is positively cooperative, displaying a sigmoidal substrate dependence curve with an [S](0.5) of 1 mM L-asparagine and a Hill coefficient (n(H)) of 2.6. Binding of L-asparagine to an allosteric site was observed in the crystal structure concomitant with a reorganization of the quarternary structure, relative to the apo enzyme. The carboxyl group of the bound asparagine makes salt bridges and hydrogen bonds to Arg240, while the N(delta2) nitrogen interacts with Thr162. Mutation of Arg240 to Ala increases the [S](0.5) value to 5.9 mM, presumably by reducing the affinity of the site for L-asparagine, although the enzyme retains cooperativity. Mutation of Thr162 to Ala results in an active enzyme with no cooperativity. Transmission of the signal from the allosteric site to the active site appears to involve subtle interactions at the dimer-dimer interface and relocation of Gln118 into the vicinity of the active site to position the probable catalytic water molecule. These data define the structural basis for the cooperative regulation of the intracellular asparaginase that is required for proper functioning within the cell. << Less

  J. Mol. Biol. 369:794-811(2007) [PubMed] [EuropePMC]

• Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid.

  Pollmann S., Neu D., Weiler E.W.

  Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohyd ... >> More

  Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against L-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1. << Less

  Phytochemistry 62:293-300(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structural characterization of Pseudomonas 7A glutaminase-asparaginase.

  Lubkowski J., Wlodawer A., Ammon H.L., Copeland T.D., Swain A.L.

  The amino acid sequence and a 2-A-resolution crystallographic structure of Pseudomonas 7A glutaminase-asparaginase (PGA) have been determined. PGA, which belongs to the family of tetrameric bacterial amidohydrolases, deamidates glutamine and asparagine. The amino acid sequence of PGA has a high de ... >> More

  The amino acid sequence and a 2-A-resolution crystallographic structure of Pseudomonas 7A glutaminase-asparaginase (PGA) have been determined. PGA, which belongs to the family of tetrameric bacterial amidohydrolases, deamidates glutamine and asparagine. The amino acid sequence of PGA has a high degree of similarity to the sequences of other members of the family. PGA has the same fold as other bacterial amidohydrolases, with the exception of the position of a 20-residue loop that forms part of the active site. In the PGA structure presented here, the active site loop is observed clearly in only one monomer, in an open position, with a conformation different from that observed for other amidohydrolases. In the other three monomers the loop is disordered and cannot be traced. This phenomenon is probably a direct consequence of a very low occupancy of product(s) of the enzymatic reaction bound in the active sites of PGA in these crystals. The active sites are composed of a rigid part and the flexible loop. The rigid part consists of the residues directly involved in the catalytic reaction as well as residues that assist in orienting the substrate. Two residues that are important for activity residue on the flexible loop. We suggest that the flexible loops actively participate in the transport of substrate and product molecules through the amidohydrolase active sites and participate in orienting the substrate molecules properly in relation to the catalytic residues. << Less

  Biochemistry 33:10257-10265(1994) [PubMed] [EuropePMC]

• The human asparaginase-like protein 1 hASRGL1 is an Ntn hydrolase with beta-aspartyl peptidase activity.

  Cantor J.R., Stone E.M., Chantranupong L., Georgiou G.

  Herein we report the bacterial expression, purification, and enzymatic characterization of the human asparaginase-like protein 1 (hASRGL1). We present evidence that hASRGL1 exhibits beta-aspartyl peptidase activity consistent with enzymes designated as plant-type asparaginases, which had thus far ... >> More

  Herein we report the bacterial expression, purification, and enzymatic characterization of the human asparaginase-like protein 1 (hASRGL1). We present evidence that hASRGL1 exhibits beta-aspartyl peptidase activity consistent with enzymes designated as plant-type asparaginases, which had thus far been found in only plants and bacteria. Similar to nonmammalian plant-type asparaginases, hASRGL1 is shown to be an Ntn hydrolase for which Thr168 serves as the essential N-terminal nucleophile for intramolecular processing and catalysis, corroborated in part by abolishment of both activities through the Thr168Ala point mutation. In light of the activity profile reported here, ASRGL1s may act synergistically with protein l-isoaspartyl methyl transferase to relieve accumulation of potentially toxic isoaspartyl peptides in mammalian brain and other tissues. << Less

  Biochemistry 48:11026-11031(2009) [PubMed] [EuropePMC]

• Uncoupling intramolecular processing and substrate hydrolysis in the N-terminal nucleophile hydrolase hASRGL1 by circular permutation.

  Li W., Cantor J.R., Yogesha S.D., Yang S., Chantranupong L., Liu J.Q., Agnello G., Georgiou G., Stone E.M., Zhang Y.

  The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. How ... >> More

  The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. However, in vitro, autoprocessing is incomplete (~50%), fettering the biophysical characterization of hASRGL1. We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled the autoprocessing reaction, allowing us to kinetically and structurally characterize this enzyme and the precursor-like hASRGL1-Thr168Ala variant. Crystallographic and biochemical evidence suggest an activation mechanism where a torsional restraint on the Thr168 side chain helps drive the intramolecular processing reaction. Cleavage and formation of the active site releases the torsional restriction on Thr168, which is facilitated by a small conserved Gly-rich loop near the active site that allows the conformational changes necessary for activation. << Less

  ACS Chem. Biol. 7:1840-1847(2012) [PubMed] [EuropePMC]

• Reactions of Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibitions and suggests an unusual catalytic triad Thr-Tyr-Glu.

  Ortlund E., Lacount M.W., Lewinski K., Lebioda L.

  Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D and L isomers of glutamine and asparagine. Crystals of PGA were reacted with diazo analogues of glutamine (6-diazo-5-oxo-L-norleucine, DON) and asparagine (5-diazo-4-oxo-L-norvaline, DONV), which are known inhibitors of th ... >> More

  Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D and L isomers of glutamine and asparagine. Crystals of PGA were reacted with diazo analogues of glutamine (6-diazo-5-oxo-L-norleucine, DON) and asparagine (5-diazo-4-oxo-L-norvaline, DONV), which are known inhibitors of the enzyme. The derivatized crystals remained isomorphous to native PGA crystals. Their structures were refined to crystallographic R = 0.20 and R(free) = 0.24 for PGA-DON and R = 0.19 and R = 0.23 for PGA-DONV. Difference Fourier electron density maps clearly showed that both DON and DONV inactivate PGA through covalent inhibition. Continuous electron density connecting the inhibitor to both Thr20 and Tyr34 of the flexible loop was observed providing strong evidence that Thr20 is the primary catalytic nucleophile and that Tyr34 plays an important role in catalysis as well. The unexpected covalent binding observed in the PGA-DON and PGA-DONV complexes shows that a secondary reaction involving the formation of a Tyr34-inhibitor bond takes place with concomitant inactivation of PGA. The predicted covalent linkage is not seen, however, suggesting an alternative method of inhibition not yet seen for these diazo analogues. These surprising results give insight as to the role of the flexible loop Thr and Tyr in the catalytic mechanism. << Less

  Biochemistry 39:1199-1204(2000) [PubMed] [EuropePMC]

• Gliap -- a novel untypical L-asparaginase localized to rat brain astrocytes.

  Dieterich D.C., Landwehr M., Reissner C., Smalla K.-H., Richter K., Wolf G., Boeckers T.M., Gundelfinger E.D., Kreutz M.R.

  L-asparaginases catalyse the formation of the neuroactive amino acid L-aspartate by deamination of asparagine. The major pathophysiological significance of L-asparaginase activity is in its clinical use for the treatment of acute lymphatic leukaemia and neoplasias that require asparagine and obtai ... >> More

  L-asparaginases catalyse the formation of the neuroactive amino acid L-aspartate by deamination of asparagine. The major pathophysiological significance of L-asparaginase activity is in its clinical use for the treatment of acute lymphatic leukaemia and neoplasias that require asparagine and obtain it from circulating pools. Here we report the identification and characterization of Gliap, a cytosolic L-asparaginase, which is the founding member of a new group of L-asparaginases in mammalia. Structural modelling suggests that Gliap is an atypical mammalian type-I asparaginase inasmuch as it harbours the active centre of a type-I glycosylasparaginase but, like plant-type asparaginases, lacks their auto-proteolytic site and, in addition, exhibits significant type-II L-asparaginase enzymatic activity. Moreover, in contrast to glycosylasparaginases Gliap is enriched in the cytosolic fraction and not in lysosomes. The protein is particularly abundant in liver, testis and brain. In brain Gliap is exclusively expressed in astrocytes and prominently present in structures reminiscent of glial endfeet. These data suggest that Gliap is involved in astroglial production of the neuroactive amino acid L-aspartate. << Less

  J. Neurochem. 85:1117-1125(2003) [PubMed] [EuropePMC]

• Structures of amidohydrolases. Amino acid sequence of a glutaminase-asparaginase from Acinetobacter glutaminasificans and preliminary crystallographic data for an asparaginase from Erwinia chrysanthemi.

  Tanaka S., Robinson E.A., Appella E., Miller M., Ammon H.L., Roberts J., Weber I.T., Wlodawer A.

  The complete amino acid sequence of a glutaminase-asparaginase from Acinetobacter glutaminasificans, for which a preliminary tertiary structure is available from crystallographic analysis, has been determined by automated Edman degradation of fragments produced by chemical and proteolytic cleavage ... >> More

  The complete amino acid sequence of a glutaminase-asparaginase from Acinetobacter glutaminasificans, for which a preliminary tertiary structure is available from crystallographic analysis, has been determined by automated Edman degradation of fragments produced by chemical and proteolytic cleavages. The protein consists of 331 amino acid residues and has a molecular weight of 35,500. The pattern of hydrophilic and hydrophobic regions is typical of a globular protein. A new crystal form of an Erwinia chrysanthemi 1125 asparaginase is reported. The space group is monoclinic C2, with unit cell parameters of: a = 107.8, b = 91.7, c = 129.2 A and beta = 91.7 degrees. A Vm of 2.25 A3/dalton was calculated for one tetramer of 35,100-dalton subunits per asymmetric unit. X-ray intensity data have been obtained to 2.2 A resolution. The point group symmetry of the Er. chrysanthemi tetramer is 222 from self-rotation function calculations. The relative orientations of an A. glutaminasificans glutaminase-asparaginase model and the Er. chrysanthemi asparaginase tetramer have been determined with the cross-rotation function, and translation function calculations have revealed a plausible location for the asparaginase tetramer in the crystal. << Less

  J. Biol. Chem. 263:8583-8591(1988) [PubMed] [EuropePMC]

• Cloning and expression of cDNA encoding rat liver 60-kDa lysophospholipase containing an asparaginase-like region and ankyrin repeat.

  Sugimoto H., Odani S., Yamashita S.

  Mammalian tissues contain small form and large form lysophospholipases. Here we report the cloning, sequence, and expression of cDNA encoding the latter form of lysophospholipase using antibody raised against the enzyme purified from rat liver supernatant (Sugimoto, H., and Yamashita, S. (1994) J. ... >> More

  Mammalian tissues contain small form and large form lysophospholipases. Here we report the cloning, sequence, and expression of cDNA encoding the latter form of lysophospholipase using antibody raised against the enzyme purified from rat liver supernatant (Sugimoto, H., and Yamashita, S. (1994) J. Biol. Chem. 269, 6252-6258). The 2,539-base pair cDNA encoded 564 amino acid residues with a calculated Mr of 60,794. The amino-terminal two-thirds of the deduced amino acid sequence significantly resembled Escherichia coli asparaginase I with the putative asparaginase catalytic triad Thr-Asp-Lys and was followed by leucine zipper motif. The carboxyl-terminal region carried ankyrin repeat. When the cDNA was transfected into HEK293 cells, not only lysophospholipase activity but also asparaginase and platelet-activating factor acetylhydrolase activities were expressed. Reverse transcription-polymerase chain reaction revealed that the transcript occurred at high levels in liver and kidney but was hardly detectable in lung and heart from which large form lysophospholipases had been purified, suggesting the presence of multiple forms of large form lysophospholipase in mammalian tissues. << Less

  J. Biol. Chem. 273:12536-12542(1998) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.