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The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate: Insights into its biological function

¹ Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA
² Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, USA

(RECEIVED February 1, 2007; FINAL REVISION March 23, 2007; ACCEPTED April 23, 2007)

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 Å 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.

Keywords: cis–trans isomerases; non-PLP-dependent isomerases protein fold; propionate catabolism; 2-methylcitric acid cycle; 2-methylcitrate dehydratase; 2-methylaconitate isomerase; aconitase; Shewanella physiology

³ These authors contributed equally to this work.

Abbreviations: 2-MC, 2-methylcitrate; 2-MCC, 2-methylcitric acid cycle; AMP, adenosine monophosphate; DMSO, dimethylsulfoxide; dNTP, deoxynucleoside triphosphate; DTT, dithiothreitol; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; RMS, root mean square; rTEV, recombinant tobacco etch virus; OD₆₀₀, optical density measured at 600 nm; PEI, polyethyleneimine; MAD phasing, multiple wavelength anomalous dispersion phasing.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072801907.

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