Temperature adaptation of DNA ligases from psychrophilic organisms

Abstract

DNA ligases operating at low temperatures have potential advantages for use in biotechnological applications. For this reason, we have characterised the temperature-optima and thermal stabilities of three minimal Lig E-type ATP-dependant DNA ligase originating from Gram-negative obligate psychrophilic bacteria. The three ligases, denoted Vib-Lig, Psy-Lig and Par-Lig show a remarkable range of thermal stabilities and optima, with the first bearing all the hallmarks of a genuinely coldadapted enzyme, while the latter two have activity and stability profiles more typical of mesophilic proteins. A comparative approach based on sequence comparison and homology modelling indicates that the cold-adapted features of Vib-Lig may be ascribed to differences in surface charge rather than increased local or global flexibility: which is consistent with the contemporary emerging paradigm of the physical basis of cold adaptation of enzymes.

Response to Cold: A Comparative Transcriptomic Analysis in Eight Cold-Adapted Yeasts

“…Several putative isozymes were found to be up- and downregulated in the same yeasts, mainly in W. anomalus or different yeasts, raising the possibility that the yeasts performed a replacement for enzymes more adequate to function at a lower temperature, as described for Saccharomyces cerevisiae when exposed to different stresses ( Ansell et al, 1997 ; Postmus et al, 2012 ). Among the structural properties reported for cold-active enzymes are an increased number of small side-chain amino acids, reduced hydrophobicity in the core of the protein, an increased number and longer random coils, reduced number of ionic and hydrogen bond interactions and larger and more flexible active sites ( Lonhienne et al, 2001 ; D’Amico et al, 2006 ; Jung et al, 2008 ; Feller, 2013 ; Santiago et al, 2016 ; Berg et al, 2019 ; Ang et al, 2021 ; Parvizpour et al, 2021 ; Yang et al, 2021 ; Yusof et al, 2021 ). When the up- vs. downregulated isozymes were compared in these properties, calculated from 3D structure models, no significant differences were found, but significant differences were found when compared to mesophyll counterparts used as a template for 3D modeling.…”

Bacteriophage origin of some minimal ATP-dependent DNA ligases: a new structure from Burkholderia pseudomallei with striking similarity to Chlorella virus ligase

“…Ligase activity with double- and single-stranded breaks were measured by denaturing urea-PAGE of fluorescently-labelled DNA duplexes as described previously 35 ; (briefly, 80 nM substrate, 0.1 mM ATP, 10 mM MgCl 2 , 1.0 mM 1,4-Dithiothreitol (DTT), 100 mM NaCl, 50 mM Tris pH 8.0) and with the following assay conditions: nicked substrate 15 min at 25 °C; cohesive overhang substrate 2 h 25 °C; mismatch substrate 2 h 15 °C, blunt and overhang substrate 2 h and 18 h 15 °C. DNA oligos used to assemble substrates are given in Supplementary Tables 4 and 5 .…”

Structural intermediates of a DNA–ligase complex illuminate the role of the catalytic metal ion and mechanism of phosphodiester bond formation

“…Ligase activity of Pmar-Lig, Ame-Lig and T4-Lig with double- and single-stranded breaks were measured by denaturing urea-PAGE of fluorescently labeled DNA duplexes as described previously (33) (briefly, 80 nM substrate, 0.1 mM ATP, 10 mM MgCl 2 , 1.0 mM 1,4-Dithiothreitol (DTT), 100 mM NaCl, 50 mM Tris pH 8.0) and with the following assay conditions: nicked substrate 15 min at 25°C; cohesive overhang substrate 2 h 25°C; blunt substrate 18 h 15°C. Metal-dependence of ligase activity by Pmar-Lig was measured by molecular beacon assay (34) with modifications described previously (22).…”