4KU3

Crystal Structure of C143S Xanthomonas Campestris OleA bound with myristic acid and myrisotoyl-CoA

Summary for 4KU3

• Entry DOI: 10.2210/pdb4ku3/pdb
• Related: 3ROW 3S21 3s23 4KT1 4ku2
• Descriptor: 3-oxoacyl-[ACP] synthase III, DI(HYDROXYETHYL)ETHER, MYRISTIC ACID, ... (5 entities in total)
• Functional Keywords: thiolase, transferase
• Biological source: Xanthomonas campestris pv. campestris
• Total number of polymer chains: 2
• Total formula weight: 79071.26

Authors

Goblirsch, B.R. (deposition date: 2013-05-21, release date: 2014-07-23, Last modification date: 2023-09-20)

Primary citation

Goblirsch, B.R.,Jensen, M.R.,Mohamed, F.A.,Wackett, L.P.,Wilmot, C.M.
Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis.
J.Biol.Chem., 291:26698-26706, 2016

PubMed Abstract: Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety-unusual for a thiolase-are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C and C) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluβ) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation.

PubMed: 27815501
DOI: 10.1074/jbc.M116.760892

Experimental method

X-RAY DIFFRACTION (1.97 Å)