RRC ID 53585
著者 Khalil S, Jaworski I, Pawelek PD.
タイトル Identification of a surface glutamine residue (Q64) of Escherichia coli EntA required for interaction with EntE.
ジャーナル Biochem Biophys Res Commun
Abstract The enterobactin biosynthetic enzyme EntA forms a complex with EntE, the next enzyme in the pathway, to enhance activation of the enterobactin precursor 2,3-dihydroxybenzoate. Here we used phage display to identify an EntE-interacting region on the surface of EntA. Upon panning immobilized EntE with a random peptide phage library, we recovered 47 unique EntE-binding dodecamer peptide sequences that aligned to a region of the EntA primary sequence corresponding to helix α4. In order to further investigate this region, we mutagenized EntA Q64, a hydrogen-bonding residue found on the surface-exposed face α4. Far-UV circular dichroism, thermal denaturation experiments, and enzymatic assays showed that mutation of EntA residue Gln 64 to alanine (Q64A) had no deleterious effect on EntA structure or function. By following near-UV CD spectral changes, we found that the spectrum of wild-type EntA was altered in the presence of EntE, indicative of conformational changes in EntA aromatic chromophores upon formation of the EntA-EntE complex. However, EntE did not affect the CD spectrum of EntA variant Q64A, demonstrating that this variant did not interact with EntE in a manner similar to wild-type EntA. Analytical ultracentrifugation of wild-type and variant EntA proteins showed that EntA Q64A was predominantly dimeric at 20μM, unlike wild-type EntA which was predominantly tetrameric. Taken together, our findings establish that EntA α4 is required for efficient formation of the EntA-EntE as well as for EntA oligomerization.
巻・号 453(3)
ページ 625-30
公開日 2014-10-24
DOI 10.1016/j.bbrc.2014.09.131
PII S0006-291X(14)01774-4
PMID 25301558
MeSH Amino Acid Sequence Circular Dichroism Escherichia coli Proteins / chemistry Escherichia coli Proteins / genetics Escherichia coli Proteins / metabolism* Glutamine / metabolism* Ligases / chemistry Ligases / genetics Ligases / metabolism* Mutagenesis, Site-Directed Oxidoreductases Acting on CH-CH Group Donors / chemistry Oxidoreductases Acting on CH-CH Group Donors / genetics Oxidoreductases Acting on CH-CH Group Donors / metabolism* Protein Binding Spectrophotometry, Ultraviolet
IF 2.985
引用数 0
リソース情報
原核生物(大腸菌)