Heterologous gene expression and characterization of TK2246, a highly active and thermostable plant type l-asparaginase from Thermococcus kodakarensis

Chohan, Shahid Mahmood; Sajed, Muhammad; Naeem, Sabeel un; Rashid, Naeem

doi:10.1016/j.ijbiomac.2020.01.012

Cited by 20 publications

(8 citation statements)

References 28 publications

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“…Phetsri et al reported maximum specific activity of 113 U/mg for Streptococcus thermophiles among four species of lactic acid bacteria tested (Phetsri et al, 2019). Specific activities of 833 and 155 U/mg are also stated for the L-asparaginases purified from Thermococcus kodakarensis and Acinetobacter soli, respectively (Jiao et al, 2020;Chohan et al, 2020).…”

Section: Selected L-asparaginase Genes Have Low Blast Similarities With the Most Used Therapeutic Enzymesmentioning

confidence: 97%

Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea

et al. 2021

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By screening 27,000 publicly available prokaryotic genomes, we recovered ca. 6300 type I and ca. 5200 type II putative L-asparaginase highlighting the vast potential of prokaryotes. Caspian water with similar salt composition to the human serum was targeted for in silico L-asparaginase screening. We screened ca. three million predicted genes of its assembled metagenomes that resulted in annotation of 87 putative L-asparaginase genes. The L-asparagine hydrolysis was experimentally confirmed by synthesizing and cloning three selected genes in E. coli. Catalytic parameters of the purified enzymes were determined to be among the most desirable reported values. Two recombinant enzymes represented remarkable anti-proliferative activity (IC50 <1IU/ml) against leukemia cell line Jurkat while no cytotoxic effect on human erythrocytes or human umbilical vein endothelial cells was detected. Similar salinity and ionic concentration of the Caspian water to the human serum highlights the potential of secretory L-asparaginases recovered from these metagenomes as potential treatment agents.

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Section: Selected L-asparaginase Genes Have Low Blast Similarities With the Most Used Therapeutic Enzymesmentioning

confidence: 97%

Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea

et al. 2021

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“…Other substitutions (Supplementary Table S2) at positions (7) Gln/ (9) Asn 0 0 0 in bacterial type 2 enzymes include (7) Gln/ (9) Ser 0 0 0 in ErAII and EwAII (both have a relatively low glutaminase co-activity; Nguyen et al, 2016;Kravchenko et al, 2008) and (7) Ser/ (9) Ser 0 0 0 in CjAII (no kinetic data). In type 1 enzymes other combinations are present: (7) Ser/ (9) Asn 0 0 0 in EcAI (no detectable glutaminase activity; Yun et al, 2007), YpAI and VcAI (no kinetic data) and (7) Thr/ (9) Gly 0 0 0 in PfAI, PhAI and TkAI (all have negligible glutaminase activity; Saeed et al, 2020;Bansal et al, 2010;Chohan et al, 2020).…”

Section: Substrate Specificity and Inhibitors Of Class 1 Asparaginasesmentioning

confidence: 99%

Structural and biophysical aspects of L-asparaginases: a growing family with amazing diversity

Loch

Jaskólski

2021

Int Union Crystallogr J

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L-Asparaginases have remained an intriguing research topic since their discovery ∼120 years ago, especially after their introduction in the 1960s as very efficient antileukemic drugs. In addition to bacterial asparaginases, which are still used to treat childhood leukemia, enzymes of plant and mammalian origin are now also known. They have all been structurally characterized by crystallography, in some cases at outstanding resolution. The structural data have also shed light on the mechanistic details of these deceptively simple enzymes. Yet, despite all this progress, no better therapeutic agents have been found to beat bacterial asparaginases. However, a new option might arise with the discovery of yet another type of asparaginase, those from symbiotic nitrogen-fixing Rhizobia, and with progress in the protein engineering of enzymes with desired properties. This review surveys the field of structural biology of L-asparaginases, focusing on the mechanistic aspects of the well established types and speculating about the potential of the new members of this amazingly diversified family.

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“…Phetsri et al, reported maximum speci c activity of 113 U/mg for Streptococcus thermophiles among four species of lactic acid bacteria tested [52]. Speci c activities of 833 and 155 U/mg are also stated for the L-asparaginases puri ed from Thermococcus Kodakarensis and Acinetobacter soli, respectively [53,54].…”

Section: Resultsmentioning

confidence: 99%

Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea

Sobat

Asad

Kabiri

et al. 2020

Preprint

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Background L-asparaginase has been used for the treatment of acute lymphoblastic leukemia (ALL) for more than 30 years. However, efforts continued to find new enzymes with more desirable properties due to the immunogenicity, short half-life, rapid clearance and L-glutaminase side activity of the existing commercial enzymes. Screening for novel L-asparaginases in prokaryotes as a promising resource has been mainly hampered by the cultivation/expression bottleneck. Results By screening 27000 publicly available prokaryotic genomes, we recovered ca. 6300 type I and ca. 5200 type II putative L-asparaginase in 36 and 42 bacterial phyla respectively highlighting the vast potential of prokaryotes for L-asparaginase activity. Caspian water with similar salt composition to the human serum was targeted for in-silico screening of L-asparaginase. We screened ca. three million predicted Open Reading Frames of the assembled Caspian Sea metagenomes. In-silico screening resulted in 87 putative L-asparaginase genes from the Caspian Sea datasets. The L-asparagine hydrolysis was experimentally confirmed by cloning three selected genes (1092, 1218 and 1011 bp) in E. coli. Catalytic parameters of the purified enzymes including Km, Vmax and catalytic efficiency were determined to be among the most desirable reported values of microbial L-asparaginases. Two of the recombinant enzymes represented remarkable anti-proliferative activity (IC50 less than 1 IU/ml) against leukemia cell line Jurkat while no cytotoxic effect on human erythrocytes or human umbilical vein endothelial cells was detected. Conclusions Similar salinity and ionic concentration of the Caspian water samples to the human serum highlights the secretory L-asparaginases recovered from these metagenomes as potential treatment agents.

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Heterologous gene expression and characterization of TK2246, a highly active and thermostable plant type l-asparaginase from Thermococcus kodakarensis

Cited by 20 publications

References 28 publications

Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea

Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea

Structural and biophysical aspects of L-asparaginases: a growing family with amazing diversity

Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea

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