Protein purification, and cloning and characterization of the cDNA and gene for xylose isomerase of barley

• Published in: European journal of biochemistry Vol. 237; no. 1; pp. 240 - 246
• Main Authors: Kristo, P, Saarelainen, R, Fagerstrom, R, Aho, S, Korhola, M
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-04-1996
• Subjects: Aldose-Ketose Isomerases; Amino Acid Sequence; barley protein; Base Sequence; Carbohydrate Epimerases - genetics; Carbohydrate Epimerases - isolation & purification; Cloning, Molecular; DNA, Complementary; genbank; genbank/x95257; Hordeum - enzymology; Hordeum - genetics; Hordeum vulgare; Hot Temperature; Hydrogen-Ion Concentration; isomerase cDNA; isomerase gene; Molecular Sequence Data; Molecular Weight; plant biochemistry; plant breeding; plant genetics; plant physiology; Sequence Homology, Amino Acid; x95256; xylose isomerase
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1996.0240n.x

The first eukaryotic xylose isomerase protein was purified from barley Hordeum vulgare. The enzyme requires Mn2+ for its activity and is fairly 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 53620 Da. A relative mass estimation of 100 000 Da was obtained from the Superose 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 nucleotides, revealed that the isomerase gene contained 20 introns, all starting with GT and ending with AG. One large intron was located in the 5' untranslated region. The barley isomerase has an insertion of about 40 residues at its amino terminus when compared to the prokaryotic cluster (family) II isomerases; cluster (family) I and cluster (family) II isomerases vary from the former in an insertion of around 50 residues at their amino termini. Comparison of the barley protein with the prokaryotic isomerases shows that the conserved catalytic and metal binding regions are also well conserved in barley.