tailieunhanh - Báo cáo Y học: Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase

Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile aminoacid residues (. N and Q) in hyperthermophilic proteins. The N þ Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperthermophiles was examined. It was found to correlate inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus licheniformis DSM13 (BLXI), which had an N þ Q content comparable to that of homologs from much more thermophilic sources. . | Eur. J. Biochem. 268 6291-6301 2001 FEBS 2001 Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase Claire Vieille1 Kevin L. Epting2 Robert M. Kelly2 and J. Gregory Zeikus1 1 Department of Biochemistry and Molecular Biology Michigan State University East Lansing MI USA 2Department of Chemical Engineering North Carolina State University Raleigh NC USA Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile aminoacid residues . 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 correlate inversely with the growth temperature of the source organism in all cases examined except for the previously uncharacterized XI from Bacillus licheniformis DSM13 BLXI which had an N Q content comparable to that of homologs from much more thermophilic sources. To determine whether BLXI behaves as a thermostable enzyme it was expressed in Escherichia coli and the thermostability and activity properties of the recombinant enzyme were studied. Indeed it was optimally active at 70-72 C which is significantly higher than the optimal growth temperature 37 C of B. licheniformis. The kinetic properties of BLXI determined at 60 C with glucose 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 temperatures of C C C and C. BLXI inactivation was first-order in all conditions examined. The energy of activation for irreversible inactivation was also strongly influenced by the metal present ranging from 342 kJ-mol 1 apoenzyme to 604 kJ-mol 1 Mg2 -enzyme to 1166