Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase

Comparative analysis of genome sequence data from mesophilic and hypertherm ophilic micro-organisms has revealed a strong bias against specific thermol abile aminoacid residues (i.e. N and Q) in hyperthermophilic proteins. The N + Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperthermophiles was examined. It was found to correlat e inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus liche niformis DSM13 (BLXI), which had an N + Q content comparable to that of hom ologs from much more thermophilic sources. To determine whether BLXI behave s as a thermostable enzyme, it was expressed in Escherichia coli, and the t hermostability and activity properties of the recombinant enzyme were studi ed. Indeed, it was optimally active at 70-72 degreesC, which is significant ly higher than the optimal growth temperature (37 degreesC) of B. lichenifo rmis. The kinetic properties of BLXI, determined at 60 degreesC with glucos e and xylose as substrates, were comparable to those of other class II XIs. The stability of BLXI was dependent on the metallic cation present in its two metal-binding sites. The enzyme thermostability increased in the order apoenzyme < Mg2+-enzyme < Co2+-enzyme = Mn2+-enzyme, with melting temperatu res of 50.3 degreesC, 53.3 degreesC, 73.4 degreesC, and 73.6 degreesC. BLXI inactivation was first-order in all conditions examined. The energy of act ivation for irreversible inactivation was also strongly influenced by the m etal present, ranging from 342 kJ.mol(-1) (apoenzyme) to 604 kJ.mol(-1) (Mg 2+-enzyme) to 1166 kJ.mol(-1) (Co2+-enzyme). These results suggest that the first irreversible event in BLXI unfolding is the release of one or both o f its metals from the active site. Although N + Q content was an indicator of thermostability for class II XIs, this pattern may not hold for other se ts of homologous enzymes. In fact, the extremely thermostable a-amylase fro m B. licheniformis was found to have an average N + Q content compared with homologous enzymes from a variety of mesophilic and thermophilic sources. Thus, it would appear that protein thermostability is a function of more co mplex molecular determinants than amino-acid content alone.