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Construction of a cysteineless variant of the E. coli pyruvate dehydrogenase E1 subunit
Author(s) -
Kale Sachin,
Nemeria Natalia,
Jordan Frank
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a46-c
Subject(s) - pyruvate dehydrogenase complex , protein subunit , pyruvate dehydrogenase phosphatase , chemistry , dihydrolipoyl transacetylase , biochemistry , enzyme , gene
The crystal structure of the E1 subunit of the pyruvate dehydrogenase complex from E. coli (PDHc‐E1) revealed three regions of low electron density, spanning residues 1–55, 401–413 and 541–557 (Arjunan et al., Biochemistry 2002 , 41, 5213–5221). Region 1–55 has been shown to interact with the PDHc‐E2 subunit (Park et al , Biochemistry 2004 , 43, 14037–14046) while region 401–413 has a role in reductive acetylation of the lipoamide of the PDHc‐E2 subunit (Nemeria et al., Biochemistry 2002 , 41, 15459–15467). Studies using site specific labeling (SSL) with thiol‐directed probes sensitive to their local environment has immense potential to characterize the dynamics of these important regions. For this purpose five of six cysteine residues in parental PDHc‐E1 (120, 259, 575, 610, 654 and 770) were converted to alanines, the sixth C259 to asparagine, by site directed mutagenesis. This PDHc‐E1 variant without cysteines showed ~4‐fold reduction in activity compared to parental PDHc‐E1 in subunit‐specific as well as overall complex activity assays. These results confirm our previous observations (Nemeria et al, Biochemistry 1998 , 37, 911–922) where none of the cysteines in singly‐substituted PDHc‐E1 were found important for activity. Moreover, reintroduction of cysteine for SSL studies in regions 1–55 (I11C) and 401–413 (Q408C) did not result in significant reduction in PDHc‐E1 activity, retaining ~40% and 80% overall activity, respectively, compared to the cysteineless PDHc‐E1 variant. These results will facilitate SSL and functional characterization of these important regions in PDHc‐E1. Supported by NIH grants GM62330 and GM53080.