Escherichia coli ilvN interacts with the FAD binding domain of ilvB and activates the AHAS I enzyme

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 H-N, N-15, C-13(alpha) and C-13(beta) atoms of the FAD binding domain of ilvB on samples that were isotopically enriched in H-2, C-13 and N-15. Unambiguous assignments were obtained for 169 of 177 backbone C-alpha atoms and 127 of 164 side chain C-beta atoms. The secondary structure determined on the basis of observed 13C(alpha) 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 ilvB alpha and ilvB beta domains was studied by both circular dichroism and isotope edited solution nuclear magnetic resonance methods. Changes in CD spectra indicate that ilvN interacts with ilvB alpha and ilvB beta domains of the catalytic subunit and not with the ilvB gamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvB and proximal to the intrasubunit ilvB alpha/ilvB beta domain interface. The implication of this interaction on the role of the regulatory subunit on the activity of the holoenzyme is discussed.