Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.

Autor: Kovermann M; Department of Chemistry, University of Konstanz, Universitätstraße, Konstanz, Germany., Stefan A; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.; CSGI, University of Firenze, Sesto Fiorentino (Firenze), Italy., Castaldo A; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy., Caramia S; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy., Hochkoeppler A; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.; CSGI, University of Firenze, Sesto Fiorentino (Firenze), Italy.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2019 Apr 10; Vol. 14 (4), pp. e0215411. Date of Electronic Publication: 2019 Apr 10 (Print Publication: 2019).
DOI: 10.1371/journal.pone.0215411
Abstrakt: 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.
Competing Interests: The authors have declared that no competing interests exist.
Databáze: MEDLINE
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