Engineering of Helicobacter pylori L-Asparaginase: Characterization of Two Functionally Distinct Groups of Mutants

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.

Glutaminase activity determines cytotoxicity of l-asparaginases on most leukemia cell lines

“…The enzyme showed a strong preference for asparagine (K m : 0.290 mM) over glutamine (K m : 46.4 mM) 21,27. A novel glutamine-inactive form of Helicobacter pylori ASNase (M121C/T169M mutant, dm HpA) was recently generated by site-directed mutagenesis 28 . The M121C mutant was designed to have the same asparaginase activity as wild type (wt), but lack glutaminase activity.…”

“…However, only with the addition of the serendipitous mutation T169M was wild-type asparaginase activity maintained in the glutaminase-free mutant. The biochemical characterization of the single and double mutant and a preliminary analysis of the cytotoxicity of the latter on HL-60 cells at 24 hours has been described elsewhere 28 .…”

The human asparaginase enzyme (ASPG) inhibits growth in leukemic cells

“…We and others [2] did not detect in ASPG any glutaminase activity, that seems to be essential for the cytotoxicity of bacterial asparaginases [18–19]. However, a mutant of Erwinia chrysanthemi L-asparaginase with very low glutaminase but relevant cytotoxic activity has been recently engineered [20].…”

Physical-Chemical Properties of L-asparaginase Mutants From Rhodospirillum Rubrum which Showed Antitelomerase Activity

“…Незначительная активность при использовании D-аспарагина и L-глутамина в качестве субстратов составляла не более 1.6% и 0.1% от L-аспарагиназной, соответственно. Есть сообщения, что снижение L-глутаминазной активности у мутантов EcA и ErA сопровождалось уменьшением L-аспарагиназной активности; у мутантов L-аспарагиназы Helicobacter pylori (HpA) L-аспарагиназная активность сохранялась, но при этом отсутствовало цитотоксическое действие на HL-60 клетки [20,21]. Низкая L-глутаминазная активность (не более 1%) известна лишь у небольшого числа бактериальных L-аспарагиназ [10,22,23,24].…”