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Modular mutagenesis of a TIM-barrel enzyme: the crystal structure of a chimeric E. coli TIM having the eighth beta alpha-unit replaced by the equivalent unit of chicken TIM
Kishan, Radha; Zeelen, Ph. Johan; Noble, Martin E.M. et al.
1994In Protein Engineering, 7 (8), p. 945-51
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Keywords :
Animals; Chickens/genetics; Crystallography; Escherichia coli/enzymology/genetics; Models, Molecular; Mutagenesis; *Protein Conformation; Protein Engineering; Recombinant Fusion Proteins/*chemistry; Triose-Phosphate Isomerase/*chemistry/genetics
Abstract :
[en] The crystal structure of a hybrid Escherichia coli triosephosphate isomerase (TIM) has been determined at 2.8 A resolution. The hybrid TIM (ETIM8CHI) was constructed by replacing the eighth beta alpha-unit of E. coli TIM with the equivalent unit of chicken TIM. This replacement involves 10 sequence changes. One of the changes concerns the mutation of a buried alanine (Ala232 in strand 8) into a phenylalanine. The ETIM8CHI structure shows that the A232F sequence change can be incorporated by a side-chain rotation of Phe224 (in helix 7). No cavities or strained dihedrals are observed in ETIM8CHI in the region near position 232, which is in agreement with the observation that ETIM8CHI and E.coli TIM have similar stabilities. The largest CA (C-alpha atom) movements, approximately 3 A, are seen for the C-terminal end of helix 8 (associated with the outward rotation of Phe224) and for the residues in the loop after helix 1 (associated with sequence changes in helix 8). From the structure it is not clear why the kcat of ETIM8CHI is 10 times lower than in wild type E.coli TIM.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Kishan, Radha;  European Molecular Biology Laboratory Germany
Zeelen, Ph. Johan;  European Molecular Biology Laboratory Germany
Noble, Martin E.M.;  European Molecular Biology Laboratory Germany
Borchert, Torben V.;  European Molecular Biology Laboratory Germany
Mainfroid, Véronique;  Université de Liège - ULiège
Goraj, Karine;  Université de Liège - ULiège
Martial, Joseph ;  Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Wierenga, Rik K.
Language :
English
Title :
Modular mutagenesis of a TIM-barrel enzyme: the crystal structure of a chimeric E. coli TIM having the eighth beta alpha-unit replaced by the equivalent unit of chicken TIM
Publication date :
1994
Journal title :
Protein Engineering
ISSN :
0269-2139
eISSN :
1460-213X
Publisher :
Oxford University Press, Oxford, United Kingdom
Volume :
7
Issue :
8
Pages :
945-51
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 25 August 2009

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