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

• Published in: PloS one Vol. 14; no. 4; p. e0215411
• Main Authors: Kovermann, Michael, Stefan, Alessandra, Castaldo, Anna, Caramia, Sara, Hochkoeppler, Alejandro
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.04.2019; Public Library of Science (PLoS)
• Subjects: Amino Acid Substitution; Biology and life sciences; Catalytic Domain; Chromatography; Diagnostic imaging; DNA; DNA Polymerase I - chemistry; DNA Polymerase I - genetics; DNA Polymerase I - metabolism; DNA polymerases; EDTA; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene expression; Genetic research; Kinetics; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Physical Sciences; Polymerase chain reaction; Protein Domains; Protein Folding; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Research and Analysis Methods; Substrate Specificity; Thermodynamics; Urea
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0215411

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.