OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis

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

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encodes the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A (CoA) molecules to form a β-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modeling, these structures suggest that Glu117 aids in substrate positioning for productive carbon-carbon bond formation. Analysis of acyl-CoA substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating that Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is affected by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer.

Original languageEnglish (US)
Pages (from-to)3871-3886
Number of pages16
JournalBiochemical Journal
Volume474
Issue number23
DOIs
StatePublished - Dec 1 2017

Bibliographical note

Funding Information:
This work was support by a grant from The BioTechnology Institute, University of Minnesota to C.M.W. and L.P. W [1000014885 10866 MNT11], National Institutes of Health (NIH) Chemistry-Biology Interface Training Grant T32GM008700 (M.R.J.), NIH Future Biotechnology Development Training Grant T32GM008347 ( J.K.C.), and University of Minnesota Doctoral Dissertation Fellowship (B.R.G.).

Funding Information:
Final X-ray diffraction data were collected at Argonne National Laboratory, GM/CA and the Structural Biology Center at the Advanced Photon Source. Argonne 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.

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