PROTEIN-PURIFICATION, AND CLONING AND CHARACTERIZATION OF THE CDNA AND GENE FOR XYLOSE ISOMERASE OF BARLEY

The first eukaryotic xylose isomerase protein was purified from barley Hordeum vulgare. The enzyme requires Mn2+ for its activity and is fai rly thermostable, with the optimum temperature being 60 degrees C. It showed maximum activity over a broad pH range (7.0-9.0). The molecular mass of the monomer was about 50000 Da based on the SDS/PAGE, and the calculated value from the cDNA-deduced polypeptide sequence was 53 62 0 Da. A relative mass estimation of 100000 Da was obtained from the Su perose 12 chromatography, suggesting that the barley enzyme is a dimer . The cloned corresponding cDNA sequence of 1710 nucleotides encoded a polypeptide of 480 amino acids. The genomic sequence of 4473 nucleoti des, revealed that the isomerase gene contained 20 introns, all starti ng with GT and ending with AG. One large intron was located in the 5'u ntranslated region. The barley isomerase has an insertion of about 40 residues at its amino terminus when compared to the prokaryotic cluste r (family) II isomerases; cluster (family) I and cluster (family) II i somerases vary from the former in an insertion of around 50 residues a t their amino termini. Comparison of the barley protein with the proka ryotic isomerases shows that the conserved catalytic and metal binding regions are also well conserved in barley.