Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose

A - Papers appearing in refereed journals

Carrier, D., van Roermund, C., Schaedler, T. A., Rong, H., Ijlist, L., Wanders, r., Baldwin, S., Waterham, H., Theodoulou, F. L. and Baker, A. 2019. Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose. Scientific Reports. 9.

AuthorsCarrier, D., van Roermund, C., Schaedler, T. A., Rong, H., Ijlist, L., Wanders, r., Baldwin, S., Waterham, H., Theodoulou, F. L. and Baker, A.
Abstract

The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of β-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed.

Year of Publication2019
JournalScientific Reports
Journal citation9
Digital Object Identifier (DOI)doi:10.1038/s41598-019-46685-9
Open accessPublished as ‘gold’ (paid) open access
Funder project or codeCleavage of acyl-CoA by peroxisomal ABC subfamily D transporters in peroxisomes: mechanism and functional roles
FunderBiotechnology and Biological Sciences Research Council
Publisher's versionCarrier_et_al-2019-Scientific_Reports (1).pdf
Output statusPublished
Publication dates
Online19 Jul 2019
Publication process dates
Accepted27 Jun 2019
PublisherNature Publishing Group
ISSN2045-2322

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