Deletion and Insertion of a 192-Residue Peptide in the Active-Site Domain of Glycosyl Hydrolase Family-2 β-Galactosidases

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概要

• 論文の詳細を見る

• The monomeric multimetal-binding β-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the "ι[iota] sequence") appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the ι sequence at specific sites of β-galactosidase on the catalytic functioning and molecular properties of β-galactosidase, deletion or insertion mutants of β-galactosidases were constructed, expressed in LacZ- E. coli strains, and characterized: srbgΔ in which the ι sequence was deleted from srbg, and lacZbgI, in which the 192-residue ι sequence was inserted into the corresponding position (between Asp^<591> and Phe^<592>) in the active-site domain of lacZbg. srbgΔ was a catalytically inactive, dimeric protein which retained multimetalbinding characteristics, suggesting that the ι sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgl existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active ( V_<max>/K_m value, 25% of that of lacZbg) and highly specific for β-D-galactoside. The tetrameric lacZbgl was activated by Mg^<2+> and Mn^<2+> with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgl, the inserted 192-residue ι peptide could fold independently of the lacZbg domains into a "sub-domain," lying distant from the active site and subunit interfaces.

• 社団法人日本生物工学会の論文
• 2002-06-20

著者

• Nishino Tokuzo

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

• Nakayama Toru

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

• 石井 正浩

  University Of Tokyo

• Nishino T

  Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University

• 石井 正治

  東京大学大学院農学生命科学研究科応用工学専攻

• Nishino Tokuzo

  Dep. Of Biochemistry And Engineering Graduate School Of Engineering Tohoku Univ. Aoba-yama Sendai Mi

• Ishii M

  Department Of Biotechnology Graduate School Of Agricultural And Life Sciences University Of Tokyo

• Ishii Masaharu

  Dep. Of Biotechnology Graduate School Of Agricultural And Life Sciences Univ. Of Tokyo

• Hemmi H

  Tohoku Univ. Miyagi Jpn

• Hemmi Hisashi

  Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University

• Hemmi Hisashi

  Department Of Biochemistry And Engineering Tohoku University

• Hemmi Hisashi

  Dep. Of Biochemistry And Engineering Graduate School Of Engineering Tohoku Univ. Aoba-yama Sendai Mi

• INOHARA-OCHIAI MISA

  Suntory Research Center

• HASEGAWA SATOSHI

  Department of Biomolecular Engineering, Graduate School of Engineering, Thhoku University

• IGUCHI SOTA

  Department of Biomolecular Engineering, Graduate School of Engineering, Thhoku University

• ASHIKARI TOSHIHIKO

  Suntory Research Center

• SHIBANO YUJI

  Suntory Research Center

• Nakayama Toru

  Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University

• Iguchi Sota

  Department Of Biomolecular Engineering Graduate School Of Engineering Thhoku University

• Shibano Yuji

  Suntory Research Center:(present Address)suntory Ltd.

• Shibano Yuji

  Department Of Molecular Biology Biomolecular Engineering Research Institute

• Shibano Yuji

  Department Of Moleular Biology Biomolecular Engineering Research Insitute

• Amachi T

  Kyoto Univ. Kyoto Jpn

• Hamada Hiroaki

  Department Of Life Science Faculty Of Science Okayama University Of Science

• Nishino Tokuzo

  Department Of Biochemistry And Engineering Faculty Of Engineering Tohoku University

• Ishii Masaharu

  Graduate School Of Agricultural And Life Sciences The University Of Tokyo

• Haruta Shin

  Department Of Biotechnology Graduate School Of Agricultural And Life Sciences The University Of Toky

• Shibano Yuji

  Suntory Limited

• Hemmi Hisashi

  Department Of Applied Molecular Biosciences Graduate School Of Bioagricultural Sciences Nagoya Unive

• Yamashita Satoshi

  Department Of Biochemistry And Engineering Graduate School Of Engineering Tohoku University

• Hasegawa S

  Department Of Biomolecular Engineering Graduate School Of Engineering Thhoku University

• NAKAYAMA Toru

  Department of Applied Microbial Technology, The Kumamoto Institute of Technology:(Present office)Institute for Fundamental Research, Suntory Ltd.

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