Adaptation to
extreme environments affects the stability and
catalytic efficiency of
enzymes, often endowing them with great industrial potential. We compared the environmental adaptation of the
secreted endonuclease I from the cold-adapted
marine fish pathogen Vibrio salmonicida (VsEndA) and the human
pathogen Vibrio cholerae (VcEndA). Kinetic analysis showed that VsEndA displayed unique halotolerance. It retained a considerable amount of activity from low concentrations to at least 0.6 m
NaCl, and was adapted to work at higher
salt concentrations than VcEndA by maintaining a low K(m) value and increasing k(cat). In
differential scanning calorimetry,
salt stabilized both
enzymes, but the effect on the calorimetric enthalpy and
cooperativity of unfolding was larger for VsEndA, indicating
salt dependence.
Mutation of
DNA binding site residues (VsEndA, Q69N and K71N; VcEndA, N69Q and N71K) affected the kinetic parameters. The VsEndA Q69N
mutation also increased the T(m) value, whereas other
mutations affected mainly DeltaH(cal). The determined
crystal structure of VcEndA N69Q revealed the loss of one
hydrogen bond present in native VcEndA, but also the formation of a new
hydrogen bond involving residue 69 that could possibly explain the similar T(m) values for native and N69Q-mutated VcEndA. Structural analysis suggested that the stability,
catalytic efficiency and
salt tolerance of EndA were controlled by small changes in the
hydrogen bonding networks and surface electrostatic potential. Our results indicate that
endonuclease I adaptation is closely coupled to the conditions of the
habitats of natural
Vibrio, with VsEndA displaying a remarkable
salt tolerance unique amongst the
endonucleases characterized so far.
