The lipoyl domain of the dihydrolipoyl succinyltransferase (E2o) component of the 2OGDH (2-oxoglutarate dehydrogenase) multienzyme complex houses the lipoic acid cofactor through covalent attachment to a specific lysine side chain residing at the tip of a β-turn. Residues within the lipoyl-lysine β-turn and a nearby prominent loop have been implicated as determinants of lipoyl domain structure and function. Protein engineering of the Escherichia coli E2o lipoyl domain (E2olip) revealed that removal of residues from the loop caused a major structural change in the protein, which rendered the domain incapable of reductive succinylation by 2-oxoglutarate decarboxylase (E1o) and reduced the lipoylation efficiency. Insertion of a new loop corresponding to that of the E. coli pyruvate dehydrogenase lipoyl domain (E2plip) restored lipoylation efficiency and the capacity to undergo reductive succinylation returned, albeit at a lower rate. Exchange of the E2olip loop sequence significantly improved the ability of the domain to be reductively acetylated by pyruvate decarboxylase (E1p), retaining approx. 10-fold more acetyl groups after 25 min than wild-type E2olip. Exchange of the β-turn residue on the N-terminal side of the E2o lipoyl-lysine DKA/V motif to the equivalent residue in E2plip (T42G), both singly and in conjunction with the loop exchange, reduced the ability of the domain to be reductively succinylated, but led to an increased capacity to be reductively acetylated by the non-cognate E1p. The T42G mutation also slightly enhanced the lipoylation rate of the domain. The surface loop is important to the structural integrity of the protein and together with Thr42 plays an important role in specifying the interaction of the lipoyl domain with its partner E1o in the E. coli 2OGDH complex.
Skip Nav Destination
Article navigation
January 2008
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
December 21 2007
The role of loop and β-turn residues as structural and functional determinants for the lipoyl domain from the Escherichia coli 2-oxoglutarate dehydrogenase complex
D. Dafydd Jones;
D. Dafydd Jones
1
*Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, U.K.
†School of Biosciences, Cardiff University, Cardiff CF10 3US, U.K.
1To whom correspondence should addressed (email jonesdd@cf.ac.uk).
Search for other works by this author on:
Richard N. Perham
Richard N. Perham
*Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, U.K.
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
August 15 2007
Revision Received:
October 05 2007
Accepted:
October 11 2007
Accepted Manuscript online:
October 11 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 409 (2): 357–366.
Article history
Received:
August 15 2007
Revision Received:
October 05 2007
Accepted:
October 11 2007
Accepted Manuscript online:
October 11 2007
Connected Content
Citation
D. Dafydd Jones, Richard N. Perham; The role of loop and β-turn residues as structural and functional determinants for the lipoyl domain from the Escherichia coli 2-oxoglutarate dehydrogenase complex. Biochem J 15 January 2008; 409 (2): 357–366. doi: https://doi.org/10.1042/BJ20071119
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.