Structure, Substrate Complexation and Reaction Mechanism of Bacterial Asparaginases

Sanches, Mário; Krauchenco, Sandra; Polikarpov, Igor

doi:10.2174/187231307779814057

Cited by 28 publications

(37 citation statements)

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“…The dimeric interface of PfA holds 2 axially opposite active sites. Earlier reports on l ‐asparaginases with 2 interfacial active sites suggest that each active site consists of catalytic triads I and II (63, 64). These triads work in concert to bring about the substrate hydrolysis and product release, respectively.…”

Section: Discussionmentioning

confidence: 97%

“…The base then activates a nucleophile that covalently binds the substrate in an acyl‐enzyme intermediate and releases ammonia (acylation). In the second step, an activated nucleophile of catalytic triad II activates a water molecule that functions as second nucleophile and causes the release of product and free enzyme (deacylation) (63). From sequence and structure analysis, it emerged that the residues for catalytic triad II are fairly conserved, whereas in triad I, Thr11 is the only conserved residue in PfA and other sequence‐related enzymes.…”

Section: Discussionmentioning

confidence: 99%

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Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

et al. 2011

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Thermophilic l-asparaginases display high stability and activity at elevated temperatures. However, they are of limited use in leukemia therapy because of their low substrate affinity and reduced activity under physiological conditions. In an attempt to combine stability with activity at physiological conditions, 3 active-site mutants of Pyrococcus furiosus l-asparaginase (PfA) were developed. The mutants, specifically K274E, showed improved enzymatic properties at physiological conditions as compared to the wild type. All variants were thermodynamically stable and resistant to proteolytic digestion. None of the enzymes displayed glutaminase activity, a highly desirable therapeutic property. All variants showed higher and significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli l-asparaginase. Our study revealed that increased substrate accessibility through the active site loop plays a major role in determining activity. A new mechanistic insight has been proposed based on molecular dynamics simulated structures, where dynamic flipping of a critical Tyr residue is responsible for the activity of thermophilic l-asparaginases. Our study not only resulted in development of PfA mutants with combination of desirable properties but also gave a mechanistic insight about their activity.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

et al. 2011

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“…Earlier reports of L-ASP crystal structures demonstrated the presence of two catalytic triads in each active site. The triad I is composed of a nucleophile (Thr), a base (Tyr) and an acidic moiety (Glu) which functions on the flexibility of the active site loop (41). A similar kind of mechanism may take place when T14 gets activated along with other two residues S117 and D92 leading to the conversion of substrate L-Asn to aspartic acid and ammonia.…”

Section: Discussionmentioning

confidence: 99%

Molecular docking study of L-Asparaginase I from Vibrio campbellii in the treatment of acute lymphoblastic leukemia (ALL)

Mohideen

2020

The EuroBiotech Journal

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The potential use of asparaginases has gained tremendous significance in the treatment of acute lymphoblastic leukemia (ALL). Earlier studies suggest L-asparaginases (L-ASP) extracted from Escherichia coli and Erwinia aroideae regulates L-asparagine (L-Asn) from the circulating blood. Prolonged exposure to these enzymes may lead to hypersensitivity reactions. So, it is important to find novel asparaginases with anti-cancer properties. The three-dimensional structure of L-ASP I from Vibrio campbellii was determined by homology modeling using EasyModeller v.4.0. The structure was validated with quality indexing tools and was deposited in Protein Model DataBase. Molecular docking was performed between L-ASP I and ligand substrate L-Asn to study enzyme-substrate interactions. Qualitative and quantitative analysis of L-ASP I enzyme was found to be reliable and stable with a significant protein quality factor (LG score: 7.129). The enzyme is a dimer, belongs to α/β class of proteins. The active sites comprises of N-glycosylation site and a catalytic triad (T14-S117-D92). The binding energy of the docked complex was calculated to be -7.45 kcal/mol. The amino acid T14 identified as a primary nucleophile essential for catalytic reaction. The enzyme L-ASP I of V. campbellii provides a detailed view of structure and functional aspects with ligand substrate L-Asn. This in silico investigation has explicitly demonstrated for the first time that cytosolic L-ASP Type I of V. campbellii to have a catalytic triad which was attributed only to periplasmic L-ASP Type II. Thus, L-ASP I can serve as anti-leukemic agent in the treatment, management and control of ALL.

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“…Повышенная чувствительность L-аспарагиназной активности RrA к присутствию даже 1 M мочевины при pH 7.4 указывает на слабые водородные связи в белке и важность его структурной организации для функционирования фермента. [33,34]: цитоплазматические ферменты 1 типа, характеризующиеся высокой K m (10 -3 М) для L-аспарагина, не оказывающие противоопухолевое действие (Bacillus subtilis, Pyrococcus horikoshii и др. [24,26]), и периплазматические 2 типа, обладающие противоопухолевой активностью и имеющие высокое сродство к аспарагину (K m 10 -5 М), например, применяемые в клинической онкологии EcA и ErA [1,32,33].…”

Section: влияние химической денатурации на стабильность вариантов Rraunclassified

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

Pokrovskaya

Александрова

Veselovsky

et al. 2019

BMCRM

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Rru_A3730 protein is a bacterial Rhodospirillum rubrum L-asparaginase (RrA), which is known by its anticancer activity. RrA variants with point amino acid substitutions in the region of 150 amino acids residues: RrA17N, K149E, RrAE149R, V150P, F151T, RrА17N, E149R, V150P, RrAE149R, V150P, showed antiproliferative properties, and also by their ability to suppress telomerase activity. This work is devoted to comparison of physical-chemical and catalytic properties of these mutant forms of RrA. It is shown that pH optimum is in the alkaline zone (8.5 – 9.3); L-glutaminase and D-asparaginase activity is respectively not more than 0.1% and 1.6% of L-asparaginase for all studied variants of RrA. The presence of the N17-terminal amino acid sequence MASMTGGQMGRGSSRQ of the capsid protein of bacteriophage T7 in the RrA structure leads to an increase in the thermal stability of mutant RrA analogues (from 50°C to 56°C) and their resistance to denaturation in the presence of 3 – 4 M urea. It is of Metal ions exhibit multidirectional effects on L-asparaginase activity of RrA. K+, Ca2+, Zn2+, Cs+, Co2+ in significantly affect the activity of L-asparaginase, while Mn2+, Cu2+, Fe3+ ions inhibit it. There was no correlation between antitelomerase (antiproliferative) activity and kinetic properties of mutant forms of L-asparaginase RrA.

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Structure, Substrate Complexation and Reaction Mechanism of Bacterial Asparaginases

Cited by 28 publications

References 0 publications

Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action

Molecular docking study of L-Asparaginase I from Vibrio campbellii in the treatment of acute lymphoblastic leukemia (ALL)

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

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