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Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

フォーマット:
論文
責任表示:
片岡, 邦重 ; Kataoka, Kunishige ; Tanizawa, Katsuyuki
言語:
英語
出版情報:
Elsevier, 2003-09-01
著者名:
掲載情報:
Journal of molecular catalysis b enzymatic
ISSN:
1381-1177  CiNii Articles  Webcat Plus  JAIRO
巻:
23
通号:
2-6
開始ページ:
299
終了ページ:
309
バージョン:
author
概要:
The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward -phenylalanine and phenylpyruvate with 1.6 and 7.8% of kcat values of the wild-type enzyme for the preferred substrates, -leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids. 続きを見る
URL:
http://hdl.handle.net/2297/1731

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