Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases

ABSTRACT

The effects of divalent metal cations on structural thermostability and the inactivation kinetics of homologous class II d-xylose isomerases (XI; EC 5.3.1.5) from mesophilic (Escherichia coli and Bacillus licheniformis), thermophilic (Thermoanaerobacterium thermosulfurigenes), and hyperthermophilic (Thermotoga neapolitana) bacteria were examined. Unlike the three less thermophilic XIs that were substantially structurally stabilized in the presence of Co²⁺ or Mn²⁺ (and Mg²⁺ to a lesser extent), the melting temperature [(T_m) ≈100 °C] of T. neapolitana XI (TNXI) varied little in the presence or absence of a single type of metal. In the presence of any two of these metals, TNXI exhibited a second melting transition between 110 °C and 114 °C. TNXI kinetic inactivation, which was non-first order, could be modeled as a two-step sequential process. TNXI inactivation in the presence of 5 mm metal at 99–100 °C was slowest in the presence of Mn²⁺[half-life (t_1/2) of 84 min], compared to Co²⁺ (t_1/2 of 14 min) and Mg²⁺ (t_1/2 of 2 min). While adding Co²⁺ to Mg²⁺ increased TNXI's t_1/2 at 99–100 °C from 2 to 7.5 min, TNXI showed no significant activity at temperatures above the first melting transition. The results reported here suggest that, unlike the other class II XIs examined, single metals are required for TNXI activity, but are not essential for its structural thermostability. The structural form corresponding to the second melting transition of TNXI in the presence of two metals is not known, but likely results from cooperative interactions between dissimilar metals in the two metal binding sites.