The crystal structure of D-xylonate dehydratase reveals functional features of enzymes from the Ilv/ED dehydratase family.

The Ilv/ED dehydratase protein family includes dihydroxy acid-, gluconate-, 6-phosphogluconate- and pentonate dehydratases. The members of this family are involved in various biosynthetic and carbohydrate metabolic pathways. Here, we describe the first crystal structure of D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) at 2.7 Å resolution and compare it with other available enzyme structures from the IlvD/EDD protein family. The quaternary structure of CcXyDHT is a tetramer, and each monomer is composed of two domains in which the N-terminal domain forms a binding site for a [2Fe-2S] cluster and a Mg2+ ion. The active site is located at the monomer-monomer interface and contains residues from both the N-terminal recognition helix and the C-terminus of the dimeric counterpart. The active site also contains a conserved Ser490, which probably acts as a base in catalysis. Importantly, the cysteines that participate in the binding and formation of the [2Fe-2S] cluster are not all conserved within the Ilv/ED dehydratase family, which suggests that some members of the IlvD/EDD family may bind different types of [Fe-S] clusters.

The Crystal Structure of a Bacterial l-Arabinonate Dehydratase Contains a [2Fe-2S] Cluster.

We present a novel crystal structure of the IlvD/EDD family enzyme, l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT, EC 4.2.1.25), which catalyzes the conversion of l-arabinonate to 2-dehydro-3-deoxy-l-arabinonate. The enzyme is a tetramer consisting of a dimer of dimers, where each monomer is composed of two domains. The active site contains a catalytically important [2Fe-2S] cluster and Mg2+ ion and is buried between two domains, and also at the dimer interface. The active site Lys129 was found to be carbamylated. Ser480 and Thr482 were shown to be essential residues for catalysis, and the S480A mutant structure showed an unexpected open conformation in which the active site was more accessible for the substrate. This structure showed the partial binding of l-arabinonate, which allowed us to suggest that the alkoxide ion form of the Ser480 side chain functions as a base and the [2Fe-2S] cluster functions as a Lewis acid in the elimination reaction.

Crystallization and X-ray diffraction analysis of an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus.

L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Šresolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, ß = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a VM value of 3.2 Å(3) Da(-1) and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Šresolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, ß = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a VM value of 4.0 Å(3) Da(-1) and a solvent content of 69%.