title: Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus.
abstract: Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn(2+) that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K(m) and dramatic decrease of k(cat), and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.
authors: Department of Bioscience and Biotechnology, Konkuk University, 1 Hayang-dong, Gangjin-gu, Seoul 143-701, Republic of Korea.
authors: Soo-Jin
authors: SJ
authors: Yeom
authors: Yeong-Su
authors: YS
authors: Kim
authors: Yu-Ri
authors: YR
authors: Lim
authors: Ki-Woong
authors: KW
authors: Jeong
authors: Jee-Young
authors: JY
authors: Lee
authors: Yangmee
authors: Y
authors: Kim
authors: Deok-Kun
authors: DK
authors: Oh
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D006863:Hydrogen-Ion Concentration; D008359:Mannose-6-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism
chemical_list: D008359:Mannose-6-Phosphate Isomerase; D015032:Zinc