Elucidation of the Specific Function of the Conserved Threonine Triad Responsible for Human l-Asparaginase Autocleavage and Substrate Hydrolysis

Nomme, J.; Su, Ying; Lavie, A.

doi:10.1016/j.jmb.2014.04.016

Cited by 34 publications

(61 citation statements)

References 34 publications

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“…Similarly, a previous study of hASRGL1 showed that T219 and T186 provide an essential hydrogen bond network that facilitates both intra-molecular processing and substrate catalysis where it was reported that T219V, T219A, and T186V variants required large amounts of glycine to achieve (partial) cleavage but displayed no detectable L-Asn hydrolysis. 3 We initiated a mutagenesis study by constructing N62D/A, T186A/S, and T219A/S active site variants to assess their role in substrate catalysis as well as processing. Using the cp-hASRGL1 platform as a way to obtain fully activated enzyme along with the use of the asparaginase substrate analogue, L-aspartic acid β -hydroxamate (AHA) provided an ideal way to detect activity of these mutated variants.…”

Section: Resultsmentioning

confidence: 99%

“…2 Furthermore, the hASRGL1 asparaginase activity has generated interest in the therapeutic development of hASRGL1 as a potentially nonimmunogenic alternative to the bacterial asparaginases used to clinically treat acute lymphoblastic leukemia. 3 …”

mentioning

confidence: 99%

“…4 The finding that free glycine (Gly) accelerates and facilitates full intramolecular cleavage of wt-hASRGL1 was another significant advance, 5 although mutational studies of active site residues have shown that glycine does not always facilitate autoprocessing, rendering kinetic quantitation difficult or impossible in certain cases. 3 Our group also previously solved this issue by creating a fully activated version of hASRGL1 by means of a circular permutation (cp-hASRGL1). This engineering design physically links the original N- and C-termini, and the protein is now expressed such that the active site nucleophile Thr168 is the new N-terminus once the initiator Met residue is cleaved during translation.…”

mentioning

confidence: 99%

“…This strategy allows the characterization of active site variants such as hASRGL1-Thr168Ser that are incapable of intramolecular processing (even in the presence of high glycine concentrations). 3,6 …”

mentioning

confidence: 99%

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Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

Irani

Crutchfield

et al. 2016

Biochemistry

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The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes L-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an αβ–βα sandwich fold homodimer that cleaves its own peptide backbone at the G167–T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167–T168 peptide bond reaches only ≤50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure–activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

Irani

Crutchfield

et al. 2016

Biochemistry

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“…ASNases are classified into two families: i.e., bacterial type or type I and type II, and plant-type or type III enzymes. The bacterial type II enzymes have been used in treating ALL because of their low micromolar K m value and relatively easier preparation, whereas the human enzyme, a type III enzyme, has a millimolar K m value for ASNase [ 25 ]. The human enzyme is poorly suitable for cancer treatment because of reduced activities.…”

Section: Targeting Specific Amino Acid Starvation In Cancer Therapmentioning

confidence: 99%

Targeting the Proline–Glutamine–Asparagine–Arginine Metabolic Axis in Amino Acid Starvation Cancer Therapy

et al. 2021

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Proline, glutamine, asparagine, and arginine are conditionally non-essential amino acids that can be produced in our body. However, they are essential for the growth of highly proliferative cells such as cancers. Many cancers express reduced levels of these amino acids and thus require import from the environment. Meanwhile, the biosynthesis of these amino acids is inter-connected but can be intervened individually through the inhibition of key enzymes of the biosynthesis of these amino acids, resulting in amino acid starvation and cell death. Amino acid starvation strategies have been in various stages of clinical applications. Targeting asparagine using asparaginase has been approved for treating acute lymphoblastic leukemia. Targeting glutamine and arginine starvations are in various stages of clinical trials, and targeting proline starvation is in preclinical development. The most important obstacle of these therapies is drug resistance, which is mostly due to reactivation of the key enzymes involved in biosynthesis of the targeted amino acids and reprogramming of compensatory survival pathways via transcriptional, epigenetic, and post-translational mechanisms. Here, we review the interactive regulatory mechanisms that control cellular levels of these amino acids for amino acid starvation therapy and how drug resistance is evolved underlying treatment failure.

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Structural and functional diversity of asparaginases: Overview and recommendations for a revised nomenclature

Silva

Pessoa

Oliveira

et al. 2021

Biotech and App Biochem

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Asparaginases (ASNases) are a large and structurally diverse group of enzymes ubiquitous amongst archaea, bacteria and eukaryotes, that catalyze hydrolysis of asparagine to aspartate and ammonia. Bacterial ASNases are important biopharmaceuticals for the treatment of acute lymphoblastic leukemia, although some patients experience adverse allergic side effects during treatment with these protein therapeutics. ASNases are currently divided into three families: plant-type ASNases, Rhizobium etli-type ASNases and bacterial-type ASNases. This system is outdated as both bacterial-type and plant-type families also include archaeal, bacterial and eukaryotic enzymes, each with their own distinct characteristics. Herein, phylogenetic studies allied to tertiary structural analyses are described with the aim of proposing a revised and more robust classification system that considers the biochemical diversity of ASNases. Accordingly, based on distinct peptide domains, phylogenetic data, structural analysis and functional characteristics, we recommend that ASNases now be divided into three new distinct classes containing subgroups according to structural and functional aspects.Using this new classification scheme, 25 ASNases were identified as candidates for future new lead discovery.

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Elucidation of the Specific Function of the Conserved Threonine Triad Responsible for Human l-Asparaginase Autocleavage and Substrate Hydrolysis

Cited by 34 publications

References 34 publications

Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages

Targeting the Proline–Glutamine–Asparagine–Arginine Metabolic Axis in Amino Acid Starvation Cancer Therapy

Structural and functional diversity of asparaginases: Overview and recommendations for a revised nomenclature

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