Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain
Autor: | Alejandro Hochkoeppler, Anna Castaldo, Alessandra Stefan, Michael Kovermann, Sara Caramia |
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Přispěvatelé: | Kovermann M., Stefan A., Castaldo A., Caramia S., Hochkoeppler A. |
Jazyk: | angličtina |
Rok vydání: | 2019 |
Předmět: |
Exonuclease
Protein Folding Stereochemistry DNA polymerase Science tryptophan fluorescence HoLaMa Substrate Specificity 03 medical and health sciences Non-competitive inhibition Protein Domains Catalytic Domain Enzyme Stability Escherichia coli DNA binding Nuclear Magnetic Resonance Biomolecular Polymerase 030304 developmental biology Klenow fragment chemistry.chemical_classification 0303 health sciences Multidisciplinary biology Chemistry Escherichia coli Proteins 030302 biochemistry & molecular biology Mutagenesis stability DNA Polymerase I NMR Peptide Fragments Recombinant Proteins Kinetics Enzyme Amino Acid Substitution ddc:540 biology.protein Mutagenesis Site-Directed Proofreading Thermodynamics Medicine |
Zdroj: | PLoS ONE, Vol 14, Iss 4, p e0215411 (2019) |
ISSN: | 1932-6203 |
Popis: | We report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3’-5’ exonuclease domain. HoLaMa was overexpressed in Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20°C. In addition, urea induced a cooperative folding to unfolding transition of HoLaMa, possessing an overall thermodynamic stability and a transition midpoint featuring ΔG and CM equal to (15.7 ± 1.9) kJ/mol and (3.5 ± 0.6) M, respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme, we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly, HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs. published |
Databáze: | OpenAIRE |
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