Abstract
Upon infection by pathogenic bacteria, production of reactive oxygen species (ROS) is part of the host organism’s first line of defence. ROS damage a number of macromolecules, and in order to withstand such a harsh environment, the bacteria need to have well-functioning ROS scavenging and repair systems. Herein, MutT is an important nucleotide-pool sanitization enzyme, which degrades 8-oxo-dGTP and thus prevents it from being incorporated into DNA. In this context, we have performed a comparative biochemical and structural analysis of MutT from the fish pathogen Aliivibrio salmonicida (AsMutT) and the human pathogen Vibrio cholerae (VcMutT), in order to analyse their function as nucleotide sanitization enzymes and also determine possible cold-adapted properties of AsMutT. The biochemical characterisation revealed that both enzymes possess activity towards the 8-oxo-dGTP substrate, and that AsMutT has a higher catalytic efficiency than VcMutT at all temperatures studied. Calculations based on the biochemical data also revealed a lower activation energy (Ea) for AsMutT compared to VcMutT, and differential scanning calorimetry experiments showed that AsMutT displayed an unexpected higher melting temperature (Tm) value than VcMutT. A comparative analysis of the crystal structure of VcMutT, determined to 2.42Å resolution, and homology models of AsMutT indicate that three unique Gly residues in loops of VcMutT, and additional long range ion-pairs in AsMutT could explain the difference in temperature stability of the two enzymes. We conclude that AsMutT is a stable, cold-active enzyme with high catalytic efficiency and reduced Ea, compared to the mesophilic VcMutT.
Highlights
When microbes attack a host, phagocytes produce reactive oxygen species (ROS) as a first line of defence [1,2,3]
A common damage generated by ROS is the Abbreviations: 8-oxoG, 8-oxo-Guanine; Nudix, nucleoside diphosphates linked to some other moiety X; RMSD, root mean square deviation; MTH1, MutT homologue 1 from human; A:T to C:G, adenine:thymine to cytosine:guanine; NMR, nuclear magnetic resonance; DSC, differential scanning calorimetry; ROS, reactive oxygen species; Ea, activation energy
Our findings show that AsMutT possesses cold-adapted properties with a higher catalytic efficiency than VcMutT mainly caused by higher kcat values, lower Km, and lower Ea due to lower activation enthalpy (DH#)
Summary
When microbes attack a host, phagocytes produce reactive oxygen species (ROS) as a first line of defence [1,2,3]. We have studied MutT from the psychrophilic fish pathogen Aliivibrio salmonicida (AsMutT) and the mesophilic human pathogen Vibrio cholerae (VcMutT) in order to obtain more information regarding their function as nucleotide sanitization enzymes and to study cold-adapted properties of AsMutT, through enzyme kinetic measurements, differential scanning calorimetry, X-ray structure determination and homology modelling. The comparative analysis of the crystal structure of VcMutT, determined to 2.42 Å resolution, and homology models of AsMutT shows that VcMutT possesses additional Gly residues in dynamic loops and AsMutT possesses additional long range ion-pairs which could explain the difference in stability We conclude that both AsMutT and VcMutT are functional MutT enzymes within their respective organisms, and that AsMutT is able to function well in its natural cold environment due to optimised substrate interactions (lower Km) and high substrate turnover (higher kcat)
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