5VXD

Crystal structure of Xanthomonas campestris OleA E117A

Summary for 5VXD

• Entry DOI: 10.2210/pdb5vxd/pdb
• Related: 5VXE 5VXF 5VXG 5VXH 5VXI
• Descriptor: 3-oxoacyl-[ACP] synthase III, GLYCEROL (3 entities in total)
• Functional Keywords: thiolase, transferase, condensation
• Biological source: Xanthomonas campestris pv. campestris (strain ATCC 33913 / DSM 3586 / NCPPB 528 / LMG 568 / P 25)
• Total number of polymer chains: 2
• Total formula weight: 77753.00

Authors

Jensen, M.R.,Wilmot, C.M. (deposition date: 2017-05-23, release date: 2017-10-25, Last modification date: 2023-10-04)

Primary citation

Jensen, M.R.,Goblirsch, B.R.,Christenson, J.K.,Esler, M.A.,Mohamed, F.A.,Wackett, L.P.,Wilmot, C.M.
OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis.
Biochem. J., 474:3871-3886, 2017

PubMed 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, encodes the enzymes necessary to produce -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 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 p 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.

PubMed: 29025976
DOI: 10.1042/BCJ20170642

Experimental method

X-RAY DIFFRACTION (1.968 Å)