Acyl carrier protein is a cellular target for the antibacterial action of the pantothenamide class of pantothenate antimetabolites.
Pantothenate is the precursor of the essential cofactor coenzyme A (CoA). Pantothenate kinase (CoaA) catalyzes the first and regulatory step in the CoA biosynthetic pathway. The pantothenate analogs N-pentylpantothenamide and N-heptylpantothenamide possess antibiotic activity against Escherichia coli. Both compounds are substrates for E. coli CoaA and competitively inhibit the phosphorylation of pantothenate. The phosphorylated pantothenamides are further converted to CoA analogs, which were previously predicted to act as inhibitors of CoA-dependent enzymes. Here we show that the mechanism for the toxicity of the pantothenamides is due to the inhibition of fatty acid biosynthesis through the formation and accumulation of the inactive acyl carrier protein (ACP), which was easily observed as a faster migrating protein using conformationally sensitive gel electrophoresis. E. coli treated with the pantothenamides lost the ability to incorporate [1-(14)C]acetate to its membrane lipids, indicative of the inhibition of fatty acid synthesis. Cellular CoA was maintained at the level sufficient for bacterial protein synthesis. Electrospray ionization time-of-flight mass spectrometry confirmed that the inactive ACP was the product of the transfer of the inactive phosphopantothenamide moiety of the CoA analog to apo-ACP, forming the ACP analog that lacks the sulfhydryl group for the attachment of acyl chains for fatty acid synthesis. Inactive ACP accumulated in pantothenamide-treated cells because of the active hydrolysis of regular ACP and the slow turnover of the inactive prosthetic group. Thus, the pantothenamides are pro-antibiotics that inhibit fatty acid synthesis and bacterial growth because of the covalent modification of ACP.
Zhang
Yong-Mei
YM
Protein Science Division, Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA. yongmei.zhang@stjude.org
Frank
Matthew W
MW
Virga
Kristopher G
KG
Lee
Richard E
RE
Rock
Charles O
CO
Jackowski
Suzanne
S
D000085:Acetates / Q000737:chemistry; D000213:Acyl Carrier Protein / Q000737:chemistry / Q000502:physiology*; D000963:Antimetabolites / Q000737:chemistry*; D001692:Biological Transport; D015153:Blotting, Western; D002850:Chromatography, Gel; D003065:Coenzyme A / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D006868:Hydrolysis; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D008956:Models, Chemical; D010205:Pantothenic Acid / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D013296:Streptococcus pneumoniae / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D000085:Acetates; D000213:Acyl Carrier Protein; D000963:Antimetabolites; D005227:Fatty Acids; D008055:Lipids; D010205:Pantothenic Acid; D003545:Cysteine; D003065:Coenzyme A