| Autor: |
Yanase, Hideshi, Maeda, Masahiro, Hagiwara, Eigo, Yagi, Hiroyuki, Taniguchi, Kenji, Okamoto, Kenji |
| Předmět: |
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| Zdroj: |
Journal of Biochemistry; 2002, Vol. 132 Issue 4, p565-572, 8p |
| Abstrakt: |
The catalytic residues of levansucrase (sucrose: 2, 6-p-D-fructan 6-β-D-fructosyltrans-ferase, EC 2.4.1.10) from Zymomonas mobilis were analyzed by random mutation and site-directed mutagenesis. We found that substitution of Glu278 with Asp and His reduced the kcat for sucrose hydrolysis 30- and 210-fold, respectively, strongly suggesting Glu278 plays a key role in catalyzing this reaction. Given the likelihood that another acidic amino residue was also involved, we constructed variants in which acidic amino acids located within homologous regions among bacterial levansucrases and fructosyl-transferases were substituted, and found that substitution of Aspl94, located in homologous region HI, abolished sucrose hydrolysis. In addition, Glu278 was determined to be situated within the DXXER motif in homologous region IV conserved among bacterial levansucrases and fructosyltransferases, while Aspl94 was within the triplet RDP motif conserved among bacterial levansucrases, fructosyltransferases and fructofuranosi-dases. Finally, comparison of our findings with published data on other site-directed mutated enzymes indicated His296, also located in homologous region IV, is crucial for catalysis of the transfructosylation reaction. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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