Visualizing the Chain-Flipping Mechanism in Fatty-Acid Biosynthesis

The acyl carrier protein (ACP) from fatty acid synthases sequesters elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. We exploited solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. The addition of a catalytic partn...

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Bibliographic Details
Published in:	Angewandte Chemie (International ed.) Vol. 53; no. 52; pp. 14456 - 14461
Main Authors:	Beld, Joris, Cang, Hu, Burkart, Michael D.
Format:	Journal Article
Language:	English
Published:	Weinheim WILEY-VCH Verlag 22.12.2014 WILEY‐VCH Verlag Wiley Wiley Subscription Services, Inc
Edition:	International ed. in English
Subjects:	acyl carrier protein Acyl Carrier Protein - chemistry Acyl Carrier Protein - metabolism BASIC BIOLOGICAL SCIENCES Biosynthesis Carriers Catalysis Catalysts chain-flipping mechanism Chains Chemistry Chemistry, Multidisciplinary E coli Elongation Escherichia coli fatty acid synthase Fatty acids Fatty Acids - biosynthesis Fluorescence Naphthalimides - chemistry Nuclear Magnetic Resonance, Biomolecular Oxadiazoles - chemistry Physical Sciences Protein Interaction Domains and Motifs Proteins Rhodamines - chemistry Science & Technology solvatochromism Solvents - chemistry DOMAIN fatty acids ESCHERICHIA-COLI chain-flipping mechanism SYNTHASE BETA-KETOACYL acyl carrier protein FLUORESCENCE SPECTROSCOPY fatty acid synthase SPINACH DYNAMICS solvatochromism
ISSN:	1433-7851, 1521-3773, 1521-3773
Online Access:	Get full text
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Summary:	The acyl carrier protein (ACP) from fatty acid synthases sequesters elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. We exploited solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. The addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, thus enhancing fluorescence to reveal the elusive chain‐flipping mechanism. This activity was confirmed by the use of a dual solvatochromic cross‐linking probe and solution‐phase NMR spectroscopy. The chain‐flipping mechanism was visualized by single‐molecule fluorescence techniques, thus demonstrating specificity between the Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII. Flipping out in style: Protein–protein interactions with the partner protein ketoacyl synthase II (KASII) cause fatty‐acid‐intermediate cargo sequestered by the acyl carrier protein (ACP) to flip from the hydrophobic core of the carrier protein into the active site of the partner protein. Solvatochromic pantetheine probes were used to visualize this event (see picture).
Bibliography:	ark:/67375/WNG-LMGQ30PL-L California Energy Commission - No. DOE DE-EE0003373 istex:F98B625171FF6CA00D98E204DDD3BBA6F5104B75 NIH - No. R01GM094924; No. R01GM095970 ArticleID:ANIE201408576 J.B. was supported by a Rubicon postdoctoral fellowship. M.D.B. and J.B. were funded by California Energy Commission CILMSF 500-10-039; DOE DE-EE0003373; NIH R01GM094924; and R01GM095970. We thank J. J. La Clair for fruitful discussions and support, X. Huang and D. J. Lee for training and support with solution protein NMR spectroscopy, and T. L. Foley (NIH) for the plasmid encoding H. sapiens ACP. J.B. was supported by a Rubicon postdoctoral fellowship. M.D.B. and J.B. were funded by California Energy Commission CILMSF 500‐10‐039; DOE DE‐EE0003373; NIH R01GM094924; and R01GM095970. We thank J. J. La Clair for fruitful discussions and support, X. Huang and D. J. Lee for training and support with solution protein NMR spectroscopy, and T. L. Foley (NIH) for the plasmid encoding ACP. H. sapiens NIH RePORTER ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 EE0003373 USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office
ISSN:	1433-7851 1521-3773 1521-3773
DOI:	10.1002/anie.201408576