The role of OleA His285 in orchestration of long-chain acyl-coenzyme A substrates

Matthew R Jensen, Brandon R. Goblirsch, Morgan A. Esler, James K Christenson, Fatuma A. Mohamed, Lawrence P Wackett, Carrie M Wilmot

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Renewable production of hydrocarbons is being pursued as a petroleum-independent source of commodity chemicals and replacement for biofuels. The bacterial biosynthesis of long-chain olefins represents one such platform. The process is initiated by OleA catalyzing the condensation of two fatty acyl-coenzyme A substrates to form a β-keto acid. Here, the mechanistic role of the conserved His285 is investigated through mutagenesis, activity assays, and X-ray crystallography. Our data demonstrate that His285 is required for product formation, influences the thiolase nucleophile Cys143 and the acyl-enzyme intermediate before and after transesterification, and orchestrates substrate coordination as a defining component of an oxyanion hole. As a consequence, His285 plays a key role in enabling a mechanistic strategy in OleA that is distinct from other thiolases.

Original languageEnglish (US)
Pages (from-to)987-998
Number of pages12
JournalFEBS Letters
Volume592
Issue number6
DOIs
StatePublished - Mar 2018

Bibliographical note

Funding Information:
This work was support by a grant from The BioTechnology Institute, University of Minnesota to CMW and LPW (1000014885 10866 MNT11), National Institutes of Health (NIH) Chemistry-Biology Interface Training Grant T32GM008700 (MRJ), NIH Future Biotechnology Development Training Grant T32GM008347 (JKC), and University of Minnesota Doctoral Dissertation Fellowship (BRG).

Funding Information:
This work was support by a grant from The BioTechnology Institute, University of Minnesota to CMW and LPW (1000014885 10866 MNT11), National Institutes of Health (NIH) Chemistry-Biology Interface Training Grant T32GM008700 (MRJ), NIH Future Biotechnology Development Training Grant T32GM008347 (JKC), and University of Minnesota Doctoral Dissertation Fellowship (BRG). Final X-ray diffraction data were collected at Argonne National Laboratory, GM/CA and the Structural Biology Center (SBC) at the Advanced Photon Source. SBC is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). We acknowledge the staff at Sectors 19 and 23 at Argonne National Laboratory Advanced Photon Source, Argonne, IL, for their technical support. Computational resources and software were made available by the Minnesota Supercomputing Institute. Preliminary X-ray diffraction data were collected at the Kahlert Structural Biology Lab, University of Minnesota, and we thank Ed Hoeffner for his technical support.

Funding Information:
Final X-ray diffraction data were collected at Argonne National Laboratory, GM/CA and the Structural Biology Center (SBC) at the Advanced Photon Source. SBC is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). We acknowledge the staff at Sectors 19 and 23 at Argonne National Laboratory Advanced Photon Source, Argonne, IL, for their technical support. Computational resources and software were made available by the Minnesota Supercomputing Institute. Preliminary X-ray diffraction data were collected at the Kahlert Structural Biology Lab, University of Minnesota, and we thank Ed Hoeffner for his technical support.

Publisher Copyright:
© 2018 Federation of European Biochemical Societies

Keywords

  • OleA
  • X-ray crystallography
  • thiolase

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