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Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.

Maggi M, Chiarelli LR, Valentini G, Scotti C - PLoS ONE (2015)

Bottom Line: Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity.The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively).These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy; Department of Biology and Biotechnologies "Lazzaro Spallanzani", Laboratory of Protein Biochemistry, University of Pavia, Pavia, Italy.

ABSTRACT
Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.

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Residue M121 and T169 localization.L-ASN, Met121 and Thr169 are represented as spheres; the subunits A (red) and C (light blue) are represented as ribbons. Thr169 locates in a loop shared by intersubunits regions and, in E. coli, it resulted to interact with residues of the near subunit in the intimate dimer. Residue Met121 locates far from the Thr169 residue. Both residues do not seem to interact with each other.
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pone.0117025.g006: Residue M121 and T169 localization.L-ASN, Met121 and Thr169 are represented as spheres; the subunits A (red) and C (light blue) are represented as ribbons. Thr169 locates in a loop shared by intersubunits regions and, in E. coli, it resulted to interact with residues of the near subunit in the intimate dimer. Residue Met121 locates far from the Thr169 residue. Both residues do not seem to interact with each other.

Mentions: Both mutants carrying each single mutation (M121C or T169M) had a reduced ASNase activity, but the double mutant had an activity quite similar to the wild type enzyme. Its preserved activity toward L-ASN most likely depends on a complementary action of the two mutations affecting residues located in two critical regions of the enzyme structure: residue Met121, near the active site region, and Thr169, in the intersubunit one (Fig. 6). The distance of Thr169 from the active site suggests the possibility to interfere with the enzyme activity even tackling regions seemingly unrelated to the catalytic mechanism.


Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.

Maggi M, Chiarelli LR, Valentini G, Scotti C - PLoS ONE (2015)

Residue M121 and T169 localization.L-ASN, Met121 and Thr169 are represented as spheres; the subunits A (red) and C (light blue) are represented as ribbons. Thr169 locates in a loop shared by intersubunits regions and, in E. coli, it resulted to interact with residues of the near subunit in the intimate dimer. Residue Met121 locates far from the Thr169 residue. Both residues do not seem to interact with each other.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321988&req=5

pone.0117025.g006: Residue M121 and T169 localization.L-ASN, Met121 and Thr169 are represented as spheres; the subunits A (red) and C (light blue) are represented as ribbons. Thr169 locates in a loop shared by intersubunits regions and, in E. coli, it resulted to interact with residues of the near subunit in the intimate dimer. Residue Met121 locates far from the Thr169 residue. Both residues do not seem to interact with each other.
Mentions: Both mutants carrying each single mutation (M121C or T169M) had a reduced ASNase activity, but the double mutant had an activity quite similar to the wild type enzyme. Its preserved activity toward L-ASN most likely depends on a complementary action of the two mutations affecting residues located in two critical regions of the enzyme structure: residue Met121, near the active site region, and Thr169, in the intersubunit one (Fig. 6). The distance of Thr169 from the active site suggests the possibility to interfere with the enzyme activity even tackling regions seemingly unrelated to the catalytic mechanism.

Bottom Line: Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity.The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively).These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Unit of Immunology and General Pathology, University of Pavia, Pavia, Italy; Department of Biology and Biotechnologies "Lazzaro Spallanzani", Laboratory of Protein Biochemistry, University of Pavia, Pavia, Italy.

ABSTRACT
Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.

Show MeSH
Related in: MedlinePlus