Critical Role of a Loop at C-Terminal Domain on the Conformational Stability and Catalytic Efficiency of Chondroitinase ABC I

Shirdel, Akram; Khalifeh, Khosrow; Golestani, Abolfazl; Ranjbar, Bijan; Khajeh, Khosro

doi:10.1007/s12033-015-9864-3

Cited by 18 publications

(10 citation statements)

References 35 publications

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“…This is supported by the high K m (or low substrate affinity) of ChABC-55-SH3 compared to the other enzymes. A similar effect was observed by Shirdel et al (29), where structurally stabilized ChABC mutants demonstrated a lower enzymatic efficiency; this was attributed to decreased flexibility of the protein since substrate binding typically results in a conformational change. Notably, increasing the number of mutations did not necessarily lead to decreased function.…”

Section: Discussionsupporting

confidence: 77%

“…Stabilization via mutagenesis often leads to decreased catalytic efficiency (k cat /K m ). For example, H700N/L701T, the most stabilizing mutation to date (29), has an aggregation temperature 16°C greater than wild-type ChABC but is 40% less catalytically efficient. In contrast, ChABC-37-SH3 has an aggregation temperature 6°C greater than wild type, is more active overall, and is only 25% less catalytically efficient for CS degradation.…”

Section: Chondroitin Sulfate a Dermatan Sulfatementioning

confidence: 99%

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Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability

Hettiaratchi

O’Meara

et al. 2020

Sci. Adv.

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Maintaining biocatalyst stability and activity is a critical challenge. Chondroitinase ABC (ChABC) has shown promise in central nervous system (CNS) regeneration, yet its therapeutic utility is severely limited by instability. We computationally reengineered ChABC by introducing 37, 55, and 92 amino acid changes using consensus design and forcefield-based optimization. All mutants were more stable than wild-type ChABC with increased aggregation temperatures between 4° and 8°C. Only ChABC with 37 mutations (ChABC-37) was more active and had a 6.5 times greater half-life than wild-type ChABC, increasing to 106 hours (4.4 days) from only 16.8 hours. ChABC-37, expressed as a fusion protein with Src homology 3 (ChABC-37-SH3), was active for 7 days when released from a hydrogel modified with SH3-binding peptides. This study demonstrates the broad opportunity to improve biocatalysts through computational engineering and sets the stage for future testing of this substantially improved protein in the treatment of debilitating CNS injuries.

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Section: Discussionsupporting

confidence: 77%

Section: Chondroitin Sulfate a Dermatan Sulfatementioning

confidence: 99%

Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability

Hettiaratchi

O’Meara

et al. 2020

Sci. Adv.

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“…Upon construction of mutants their structural properties and biochemical features were evaluated and discussed. In spite of our previous study [10] both mutants of current research are stabilized in comparison with wild type (WT) protein.…”

Section: Introductioncontrasting

confidence: 67%

“…In this study, two single mutants (L679S, L679D) were constructed. Expression and Purification of WT and mutant proteins were carried out as previously described and protein concentration was determined by the Bradford method .…”

Section: Methodsmentioning

confidence: 99%

“…We previously found that the loop containing residues 681–695 located at C‐terminal domain of the enzyme can affect the stability of the folded state of cABC Ι. We also found that the activity of enzyme in more stabilized mutants decreases, while it increases in destabilized variants . Here, we investigated the effect of a short‐length helix at C‐terminal domain of the enzyme on the conformational stability and catalytic efficiency of cABC I.…”

Section: Introductionmentioning

confidence: 94%

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Optimization of conformational stability and catalytic efficiency in chondroitinase ABC Ι by protein engineering methods

Shamsi

Shirdel

Jafarian

et al. 2016

Engineering in Life Sciences

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Chondroitinase ABC Ι can promote the recovery of spinal cord injuries by depolimerization of glycosaminoglycans. However, low thermal stability is one of the limitations regarding its clinical application. In order to increase the conformational stability of the enzyme, Leu679 at the starting point of a short helix located at the C‐terminal domain of the protein was replaced by serine (L679S mutant) and aspartic acid (L679D mutant). Theoretical and spectroscopic studies showed that the stability of enzyme increased upon mutation. Based on the activity measurements, the catalytic efficiency of L679S was improved in comparison with the wild‐type protein; while that of L679D (a more stabilized protein) was not changed. According to the structural and kinetic data, we proposed a model in which a higher conformational stability results in a slower rate of the formation of the open conformation. On the other hand, a higher flexibility slows down the rate of the formation and holding of the closed conformation. Therefore, the L679S mutant, which is structurally stable relative to the wild‐type protein and is destabilized compared to the L679D mutant, exhibited the best catalytic efficiency. However, it was also found that the L679D mutant was more suitable for long‐term storage of the enzyme.

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Engineering a thermostable chondroitinase for production of specifically distributed low‐molecular‐weight chondroitin sulfate

Wang

Zhang

Wang

et al. 2021

Biotechnology Journal

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Chondroitinase ABC I (csABC I) has attracted intensive attention because of its great potential in heparin refining and the enzymatic preparation of low-molecular-weight chondroitin sulfate (LMW-CS). However, low thermal resistance (<30°C) restricts its applications. Herein, structure-guided and sequence-assisted combinatorial engineering approaches were applied to improve the thermal resistance of Proteus vulgaris csABC I. By integrating the deletion of the flexible fragment R166-L170 at the N-terminal domain and the mutation of E694P at the C-terminal domain, variant NΔ5/E694P exhibited 247-fold improvement of its half-life at 37°C and a 2.3-fold increase in the specific activity. Through batch fermentation in a 3-L fermenter, the expression of variant NΔ5/E694P in an Escherichia coli host reached 1.7 g L −1 with the activity of 1.0 × 10 5 U L −1 . Finally, the enzymatic approach for the preparation of LMW-CS was established. By modulating enzyme concentration and controlling depolymerization time, specifically distributed LMW-CS (7000, 3400, and 1900 Da) with low polydispersity was produced, demonstrating the applicability of these processes for the industrial production of LMW-CS in a more environmentally friendly way.

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Critical Role of a Loop at C-Terminal Domain on the Conformational Stability and Catalytic Efficiency of Chondroitinase ABC I

Cited by 18 publications

References 35 publications

Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability

Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability

Optimization of conformational stability and catalytic efficiency in chondroitinase ABC Ι by protein engineering methods

Engineering a thermostable chondroitinase for production of specifically distributed low‐molecular‐weight chondroitin sulfate

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