Solution Structure of 4鈥?Phosphopantetheine - GmACP3 from Geobacter metallireducens: A Specialized Acyl Carrier Protein with Atypical Structural Features and a Putative Role in Lipopolysacchari

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• 作者：Theresa A. Ramelot ; Matthew J. Smola ; Hsiau-Wei Lee ; Colleen Ciccosanti ; Keith Hamilton ; Thomas B. Acton ; Rong Xiao ; John K. Everett ; James H. Prestegard ; Gaetano T. Montelione ; Michael A. Kennedy
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：March 8, 2011
• 年：2011
• 卷：50
• 期：9
• 页码：1442-1453
• 全文大小：1113K
• 年卷期：v.50,no.9(March 8, 2011)
• ISSN：1520-4995

文摘

GmACP3 from Geobacter metallireducens is a specialized acyl carrier protein (ACP) whose gene, gmet_2339, is located near genes encoding many proteins involved in lipopolysaccharide (LPS) biosynthesis, indicating a likely function for GmACP3 in LPS production. By overexpression in Escherichia coli, about 50% holo-GmACP3 and 50% apo-GmACP3 were obtained. Apo-GmACP3 exhibited slow precipitation and non-monomeric behavior by ¹⁵N NMR relaxation measurements. Addition of 4鈥?phosphopantetheine (4鈥?PP) via enzymatic conversion by E. coli holo-ACP synthase resulted in stable >95% holo-GmACP3 that was characterized as monomeric by ¹⁵N relaxation measurements and had no indication of conformational exchange. We have determined a high-resolution solution structure of holo-GmACP3 by standard NMR methods, including refinement with two sets of NH residual dipolar couplings, allowing for a detailed structural analysis of the interactions between 4鈥?PP and GmACP3. Whereas the overall four helix bundle topology is similar to previously solved ACP structures, this structure has unique characteristics, including an ordered 4鈥?PP conformation that places the thiol at the entrance to a central hydrophobic cavity near a conserved hydrogen-bonded Trp-His pair. These residues are part of a conserved WDSLxH/N motif found in GmACP3 and its orthologs. The helix locations and the large hydrophobic cavity are more similar to medium- and long-chain acyl-ACPs than to other apo- and holo-ACP structures. Taken together, structural characterization along with bioinformatic analysis of nearby genes suggests that GmACP3 is involved in lipid A acylation, possibly by atypical long-chain hydroxy fatty acids, and potentially is involved in synthesis of secondary metabolites.