Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

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• 作者：Soo-Jin  ; Yeom ; Yeong-Su  ; Kim ; Yu-Ri  ; Lim ; Ki-Woong  ; Jeong ; Jee-Young  ; Lee ; Yangmee  ; Kim ; Deok-Kun  ; Oh  ; ;   ; ^{deokkun@konkuk.ac.kr}
• 关键词：Mannose-6-phosphate isomerase ; Thermus thermophilus ; Characterization ; Active-site residues
• 刊名：Biochimie
• 出版年：2011
• 出版时间：October 2011
• 年：2011
• 卷：93
• 期：10
• 页码：1659-1667
• 全文大小：914 K

文摘

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?¡ãC in the presence of 0.5?mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85?¡ãC were 13, 6.5, 3.7, 1.8, and 0.2?h, respectively. The 15 putative active-site residues within 4.5?? 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?? 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?? from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.