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

Abstract

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 In nature, short-chain fatty acids (e.g., acetate, propionate, butyrate) are very abundant (>0.1 M) in environments such as soil and the human intestine, and are of great relevance to agriculture and human health and nutrition (Cummings et al. 1987;Cummings 1995;Buckel 1999 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-N9-2-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; RMS, root mean square; rTEV, recombinant tobacco etch virus; OD 600 , 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

Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis

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Crystallization and preliminary X-ray diffraction analysis of diaminopimelate epimerase fromEscherichia coli

“…The enzyme is a symmetrical monomer comprised of two domains, each containing eight -strands and two -helices. This unique fold was first observed in H. influenzae DAP epimerase; since then, a number of proteins including a phenazine-biosynthetic protein (Blankenfeldt et al, 2004;Parsons et al, 2004), a proline racemase (Buschiazzo et al, 2006) and a methylaconitate isomerase (Garvey et al, 2007) have also been shown to adopt this fold.…”

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

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