XYLA CLONING AND SEQUENCING AND BIOCHEMICAL-CHARACTERIZATION OF XYLOSE ISOMERASE FROM THERMOTOGA-NEAPOLITANA

The xylA gene coding for xylose isomerase from the hyperthermophile Th ermotoga neapolitana 5068 was cloned, sequenced, and expressed in Esch erichia coli. The gene encoded a polypeptide of 444 residues with a ca lculated molecular weight of 50,892. The native enzyme was a homotetra mer with a molecular weight of 200,000. This xylose isomerase was a me mber of the family II enzymes (these differ from family I isomerases b y the presence of approximately 50 additional residues at the amino te rminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The cat alytic efficiency (k(cat)/K-m) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased be tween 90 and 98 degrees C primarily because of a large increase in K-m . The T. neapolitana xylose isomerase had a higher turnover number and a lower K-m for glucose than other family II xylose isomerases. Compa risons with other xylose isomerases showed that the catalytic and cati on binding regions were,yell conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine r esidues decreased with increasing enzyme thermostability, presumably a s a thermophilic strategy for diminishing the potential for chemical d enaturation through deamidation at elevated temperatures.