Crystal Structure of an Intramolecular Mesaconyl-Coenzyme A Transferase From the 3-Hydroxypropionic Acid Cycle of Roseiflexus castenholzii

Abstract

Coenzyme A (CoA) transferases catalyze reversible transfer of CoA groups from CoA-thioesters to free acids, playing important roles in the metabolism of carboxylic acids in all organisms. An intramolecular CoA transferase, Mesaconyl-CoA C1-C4 CoA transferase (MCT) was identified in the autotrophic CO2 fixation pathway, 3-hydroxypropionic acid cycle of filamentous anoxygenic phototrophs (FAPs). Different from the well-known CoA transferases that catalyze CoA transfer between two distinct substrates, MCT specifically catalyzes the reversible transformation of mesaconyl-C1-CoA to mesaconyl-C4-CoA, a key reaction intermediate for carbon fixation. However, the molecular mechanism of MCT in employing one substrate is enigmatic. Here we determined the crystal structure of MCT from a chlorosome-less FAP Roseiflexus castenholzii at 2.5 Å resolution, and characterized the catalytic mechanisms through structural analyses and molecular dynamic simulations. The structure of R. castenholzii MCT consists of a Rossmann fold larger domain and a small domain that are connected by two linkers. Two MCT subunits are cross interlocked at the linker regions to form a functional dimer in solution, in which the substrate binding pockets are located at the interface of the Rossmann fold larger domain from one subunit and the small domain from the other subunit. In the simulated binding structures, both the substrate mesaconyl-C1-CoA and product mesaconyl-C4-CoA form extensive electrostatic and hydrogen bonding interactions with MCT. But some differences exist in the binding mode of these two CoA analogs, Arg314’ from the second subunit of the dimer presenting dramatic conformational changes in binding with mesaconyl-C4-CoA. Together with Arg47 and one water molecule, a strictly conserved residue Asp165 are essential for catalyzing the reversible intramolecular CoA transfer reaction, through the electrostatic and hydrogen bonding interactions with the mesaconic tail of both the substrate and product. This study revealed a previously unrecognized mechanism for the uncommon intramolecular CoA transfer reaction, which will not only broaden the knowledge on the catalytic mechanisms of CoA transferases, but also contribute to enzyme engineering or biosynthetic applications of the 3-HP cycle for synthesis of fine chemicals and important metabolites.

Structural Basis for a Cork-Up Mechanism of the Intra-Molecular Mesaconyl-CoA Transferase

“…Since there is no need to introduce an additional substrate during the catalytic cycle, it has been speculated that the active site stays fully closed during catalysis. 1,21 This hypothesis is consistent with the observation that small inactivating molecules that could react with the acyl-enzyme anhydride intermediate, such as hydroxylamine or borohydride, had little or even no effect on Mct activity. 1 However, this also means that mesaconate would need to re-orient within the active site of Mct to enable CoA transfer from C1 to C4.…”

“…aurantiacus, R. castenholzii, Candidatus Accumulibacter phosphatis, and the γ1-endosymbiont of the gutless worm Olavius algarvensis . Overall, the tight binding of CoA along the substrate tunnel together with kinking of the adenine prevents trapped molecules from escaping and other molecules from entering the active site (Figure B–E), effectively sealing the catalytic site in a “cork-like” fashion.…”

“…Based on the structure of the apoenzyme, molecular dynamics simulations with mesaconyl-C1- and C4-CoA were performed and a mechanism for the intra-molecular CoA transfer of Mct was proposed, which differed from the canonical family III/Frc family CoA transferases. Notably, a direct, water-assisted attack of the free CoAS – onto the free carboxy group of mesaconate has been postulated . However, this mechanism seems biochemically infeasible and support for this mechanism is lacking.…”

“…Recently, the structure of the Mct homologue from Roseiflexus castenholzii (PDB 7XKG) was reported in its apo form . This structure showed that a flexible glycine-rich loop that supposedly gates catalysis in some other inter-molecular family III/Frc family CoA transferases , is absent in Mct, indicating that the reaction may proceed differently in the intra-molecular CoA transferases.…”

“…This structure showed that a flexible glycine-rich loop that supposedly gates catalysis in some other inter-molecular family III/Frc family CoA transferases , is absent in Mct, indicating that the reaction may proceed differently in the intra-molecular CoA transferases. Based on the structure of the apoenzyme, molecular dynamics simulations with mesaconyl-C1- and C4-CoA were performed and a mechanism for the intra-molecular CoA transfer of Mct was proposed, which differed from the canonical family III/Frc family CoA transferases. Notably, a direct, water-assisted attack of the free CoAS – onto the free carboxy group of mesaconate has been postulated .…”