Escherichia coli ilvN Interacts with the FAD Binding Domain of ilvB and Activates the AHAS I Enzyme

Mitra, Ashima and Sarma, Siddhartha P (2008) Escherichia coli ilvN Interacts with the FAD Binding Domain of ilvB and Activates the AHAS I Enzyme. In: Biochemistry, 47 (6). pp. 1518-1521.

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Abstract

The unique multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been exploited to generate polypeptide fragments which, when cloned and expressed, reassemble in the presence of cofactors to yield a catalytically competent enzyme. Multidimensional multinuclear NMR methods have been employed for obtaining near complete sequence specific NMR assignments for backbone HN, 15N, 13Cα and 13Cβ atoms of the FAD binding domain of ilvB on samples that were isotopically enriched in 2H, 13C and 15N. Unambiguous assignments were obtained for 169 of 177 backbone Cα atoms and 127 of 164 side chain Cβ atoms. The secondary structure determined on the basis of observed 13Cα secondary chemical shifts and sequential NOEs agrees well with the structure of this domain in the catalytic subunit of yeast AHAS. Binding of ilvN to the ilvBα and ilvBβ domains was studied by both circular dichroism and isotope edited solution nuclear magnetic resonance methods. Changes in CD spectra indicate that ilvN interacts with ilvBα and ilvBβ domains of the catalytic subunit and not with the ilvBγ domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvBβ and proximal to the intrasubunit ilvBα/ilvBβ domain interface. The implication of this interaction on the role of the regulatory subunit on the activity of the holoenzyme is discussed.

Item Type:	Journal Article
Publication:	Biochemistry
Publisher:	American Chemical Society
Additional Information:	Copyright of this article belongs to American Chemical Society.
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	17 Dec 2008 14:36
Last Modified:	19 Sep 2010 04:54
URI:	http://eprints.iisc.ac.in/id/eprint/16873

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