In bacteria, the
dehydration of
2-methylcitrate to yield 2-methylaconitate in the
2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD)
enzyme or by an
aconitase-like (AcnD)
enzyme. Bacteria that use AcnD also require the function of the PrpF
protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF
protein from the bacterium Shewanella oneidensis was solved at 2.0 A resolution. The
protein fold of PrpF is strikingly similar to those of the non-PLP-dependent
diaminopimelate epimerase from Haemophilus influenzae, a putative
proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical
protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-
aconitate to cis-
aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed
proton abstraction coupled with a rotation about C2-C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent
isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of
aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of
aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the
isomerase activity of PrpF may now be viewed as an integral part of the
2-methylcitric acid cycle.