Identification and Structural Analysis of an l-Asparaginase Enzyme from Guinea Pig with Putative Tumor Cell Killing Properties

Schalk, Amanda M.; Nguyen, Hien Anh; Rigouin, Coraline; Lavie, A.

doi:10.1074/jbc.m114.609552

Cited by 59 publications

(83 citation statements)

References 40 publications

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“…Gene Cloning and Mutagenesis-A codon-optimized synthetic gene corresponding to the amino acid sequence of ErA (UniProt entry P06608) lacking the first 21-amino acid signal peptide was synthesized by Genscript as described earlier (29). The synthetic gene was digested with NdeI and BamHI-HF restriction enzymes, gel-purified, and ligated into a His 6 -SUMOpET14b vector (where the His 6 tag is followed by the yeast protein SUMO (small ubiquitin modifier, Smt3p) using Instant Sticky End DNA ligase (New England BioLabs), generating the His 6 -SUMO-pET14b-ErA-WT plasmid.…”

Section: Methodsmentioning

confidence: 99%

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Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

Nguyen

Lavie

2016

Journal of Biological Chemistry

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Current FDA-approved L-asparaginases also possess significant L-glutaminase activity, which correlates with many of the toxic side effects of these drugs. Therefore, L-asparaginases with reduced L-glutaminase activity are predicted to be safer. We exploited our recently described structures of the Erwinia chrysanthemi L-asparaginase (ErA) to inform the design of mutants with diminished ability to hydrolyze L-glutamine. Structural analysis of these variants provides insight into the molecular basis for the increased L-asparagine specificity. A primary role is attributed to the E63Q mutation that acts to hinder the correct positioning of L-glutamine but not L-asparagine. The substitution of Ser-254 with either an asparagine or a glutamine increases the L-asparagine specificity but only when combined with the E63Q mutation. The A31I mutation reduces the substrate K m value; this is a key property to allow the required therapeutic L-asparagine depletion. Significantly, an ultra-low L-glutaminase ErA variant maintained its cell killing ability. By diminishing the L-glutaminase activity of these highly active L-asparaginases, our engineered ErA variants hold promise as L-asparaginases with fewer side effects.L-Asparaginases are amidohydrolases that catalyze the hydrolysis of the amino acid L-asparagine (Asn) 2 to L-aspartate (Asp) and ammonia. Currently, the Food and Drug Administration has approved the L-asparaginase from Escherichia coli (EcA) and Erwinia chrysanthemi (ErA) for treatment of select blood cancers. These enzyme drugs function by depleting Asn from the blood, thereby affecting certain blood cancers that are dependent on exogenous Asn. Sensitivity to L-asparaginase has been attributed to a reduced ability of those cancers to synthesize this amino acid de novo (1). Because the concentration of Asn in human blood is ϳ50 M (2), for an L-asparaginase to be clinically useful, its Asn K m value must be in the low micromolar range. This property is present in EcA (K m ϭ 15 M) and ErAPatients treated with L-asparaginase often confront two types of adverse effects. The first is due to an immune response against the bacterial enzymes. To decrease the immunogenicity, the current standard of care in the United States employs a PEGylated version of EcA (3, 4). If immunogenicity still arises, ErA is used as second line treatment (5).The second source of adverse effects of L-asparaginase treatment relates to the enzyme's ability to hydrolyze the amino acid L-glutamine (Glu). That is, in addition to catalyzing the hydrolysis of Asn (L-asparaginase activity), both EcA and ErA also catalyze the hydrolysis of Gln to L-glutamate (Glu) and ammonia (L-glutaminase activity). L-Glutamine is the most abundant amino acid in the blood with a concentration range of 400 -650 M (2). The L-glutaminase activity of these bacterial L-asparaginases is significant, ϳ2% of the EcA activity and as high as 10% for ErA (3). Of note, the L-glutaminase activity of the clinically used L-asparaginases has been implicated in many of the side effe...

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Section: Methodsmentioning

confidence: 99%

“…Protein Expression and Purification-Protein expression and purification were performed as previously described (29). In brief, plasmids corresponding to wild-type ErA (ErA-WT) or the mutant constructs (verified by sequencing) were transformed into E. coli BL21(DE3) C41 cells for expression.…”

Section: Methodsmentioning

confidence: 99%

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

Nguyen

Lavie

2016

Journal of Biological Chemistry

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“…We have recently reported the first structural characterization of a mammalian type I/II L-asparaginase, that from the guinea pig (5). By sequence homology, this guinea pig L-asparaginase would be classified as a type I enzyme; therefore we refer to it as gpASNase1.…”

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confidence: 99%

“…Furthermore, treatment efficacy can be greatly compromised upon the development of antibodies, which inactivate and deplete the enzyme. Because gpASNase1 is a mammalian L-asparaginase, it would be expected to have lower immunogenicity compared with the Food and Drug Administration-approved bacterial type II enzymes; therefore we suggested that the guinea pig enzyme would make a promising replacement for the bacterial enzymes (5).…”

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confidence: 99%

“…The design criteria for an enzyme that could potentially replace the bacterial L-asparaginases in the clinic are low micromolar K m value concomitant with high turnover rate (these two criteria will assure complete and rapid depletion of blood Asn levels). Previously we identified the guinea pig L-asparaginase that has the required low K m property, albeit in the upper range of the desired value, and reported its crystal structure with and without the product Asp (5). A more clinically relevant variant would have even a lower K m and an increased catalytic rate.…”

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Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Schalk

Antansijevic

Caffrey

et al. 2016

Journal of Biological Chemistry

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Bacterial L-asparaginases play an important role in the treatment of certain types of blood cancers. We are exploring the guinea pig L-asparaginase (gpASNase1) as a potential replacement of the immunogenic bacterial enzymes. The exact mechanism used by L-asparaginases to catalyze the hydrolysis of asparagine into aspartic acid and ammonia has been recently put into question. Earlier experimental data suggested that the reaction proceeds via a covalent intermediate using a ping-pong mechanism, whereas recent computational work advocates the direct displacement of the amine by an activated water. To shed light on this controversy, we generated gpASNase1 mutants of conserved active site residues (T19A, T116A, T19A/T116A, K188M, and Y308F) suspected to play a role in hydrolysis. Using x-ray crystallography, we determined the crystal structures of the T19A, T116A, and K188M mutants soaked in asparagine. We also characterized their steady-state kinetic properties and analyzed the conversion of asparagine to aspartate using NMR. Our structures reveal bound asparagine in the active site that has unambiguously not formed a covalent intermediate. Kinetic and NMR assays detect significant residual activity for all of the mutants. Furthermore, no burst of ammonia production was observed that would indicate covalent intermediate formation and the presence of a ping-pong mechanism. Hence, despite using a variety of techniques, we were unable to obtain experimental evidence that would support the formation of a covalent intermediate. Consequently, our observations support a direct displacement rather than a ping-pong mechanism for L-asparaginases.Currently, there still exists a gap in knowledge regarding the enzymatic mechanism by which L-asparaginases hydrolyze the amino acid L-asparagine to L-aspartic acid and ammonia (Fig. 1A). L-Asparaginases have garnered special attention because of their clinical use in the treatment of certain blood cancers (1). In addition, L-asparaginases are also used commercially by the food industry for reducing the production of acrylamide (2), a compound being investigated for its toxicity and carcinogenicity in humans. Hence, understanding the enzymatic mechanism of these important enzymes can be used to inform the design of variants with improved properties. Our specific interest is with the guinea pig L-asparaginase type I enzyme, which holds potential as a replacement for the currently clinically used bacterial enzymes (see more below).L-Asparaginases can be divided, based on sequence and structural homology, into two unrelated families: type I and II L-asparaginases belong to one family, and type III enzymes belong to another (3). The type I and II enzymes are very similar in terms of overall structure and conservation of active site residues (3). In Escherichia coli, the type I enzyme is cytoplasmic, and type II is periplasmic (3). In addition to having different cellular localization, bacterial type II enzymes have a much lower K m value for Asn (low micromolar versus millimolar). This ϳ...

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Glutaminases

Márquez

Matés

Campos-Sandoval

2016

Advances in Neurobiology

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Mammalian glutaminases catalyze the stoichiometric conversion of L-glutamine to L-glutamate and ammonium ions. In brain, glutaminase is considered the prevailing pathway for synthesis of the neurotransmitter pool of glutamate. Besides neurotransmission, the products of glutaminase reaction also fulfill crucial roles in energy and metabolic homeostasis in mammalian brain. In the last years, new functional roles for brain glutaminases are being uncovered by using functional genomic and proteomic approaches. Glutaminases may act as multifunctional proteins able to perform different tasks: the discovery of multiple transcript variants in neurons and glial cells, novel extramitochondrial localizations, and isoform-specific proteininteracting partners strongly support possible moonlighting functions for these proteins. In this chapter, we present a critical account of essential works on brain glutaminase 80 years after its discovery. We will highlight the impact of recent findings and thoughts in the context of the glutamate/glutamine brain homeostasis.

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Identification and Structural Analysis of an l-Asparaginase Enzyme from Guinea Pig with Putative Tumor Cell Killing Properties

Cited by 59 publications

References 40 publications

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Glutaminases

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