The FucO protein, a member of the group III "iron-activated" dehydrogenases, catalyzes the interconversion between L-lactaldehyde and L-1,2-propanediol in Escherichia coli. The three-dimensional structure of FucO in a complex with NAD ؉ was solved, and the presence of iron in the crystals was confirmed by X-ray fluorescence. The FucO structure presented here is the first structure for a member of the group III bacterial dehydrogenases shown experimentally to contain iron. FucO forms a dimer, in which each monomer folds into an ␣/␤ dinucleotide-binding N-terminal domain and an all-␣-helix C-terminal domain that are separated by a deep cleft. The dimer is formed by the swapping (between monomers) of the first chain of the ␤-sheet. The binding site for Fe 2؉ is located at the face of the cleft formed by the C-terminal domain, where the metal ion is tetrahedrally coordinated by three histidine residues (His200, His263, and His277) and an aspartate residue (Asp196). The glycine-rich turn formed by residues 96 to 98 and the following ␣-helix is part of the NAD ؉ recognition locus common in dehydrogenases. Site-directed mutagenesis and enzyme kinetic assays were performed to assess the role of different residues in metal, cofactor, and substrate binding. In contrast to previous assumptions, the essential His267 residue does not interact with the metal ion. Asp39 appears to be the key residue for discriminating against NADP ؉ . Modeling L-1,2-propanediol in the active center resulted in a close approach of the C-1 hydroxyl of the substrate to C-4 of the nicotinamide ring, implying that there is a typical metal-dependent dehydrogenation catalytic mechanism.In Escherichia coli and other enterobacteria the anaerobic metabolism of L-fucose and L-rhamnose requires the enzyme lactaldehyde:propanediol oxidoreductase (FucO), which is encoded by the fucO gene of the fucose regulon (6,14,16,24,32). The breakdown of these methylpentoses generates the intermediate metabolite L-lactaldehyde, which under anaerobic conditions, with NADH as a cofactor, is reduced by FucO to L-1,2-propanediol, which is excreted as a fermentation product (14). In mutant strains of E. coli adapted to grow on L-1,2-propanediol, FucO catalyzes the oxidation of the polyol to L-lactaldehyde, which is subsequently oxidized to L-lactate by a specific aldehyde dehydrogenase (41) and introduced into the general metabolism. FucO, which is induced regardless of the respiratory conditions of the culture, remains fully active in the absence of oxygen (11). In the presence of oxygen, this enzyme becomes oxidatively inactivated by a metal-catalyzed oxidation mechanism (10).FucO is an iron-dependent metalloenzyme that is inactivated by other metals, such as zinc, copper, or cadmium (40), and has been reported to be a homodimer formed by monomers consisting of 383 amino acids and having a molecular mass of 40,644 Da. The iron in the active center accounts for the oxidative inactivation of FucO mentioned above (10). A putative iron-binding motif encompassing a 15-amino-ac...

No abstract

The anaerobic utilization of L-ascorbate by gene products of the ula regulon in Escherichia coli has been widely documented. Under aerobic conditions, we have shown that this metabolism is only functional in the presence of casein acid hydrolysate. Transcriptional fusions and proteomic analysis indicated that both the ula regulon and the yiaK-S operon are required for the aerobic utilization of this compound. The aerobic dissimilation of L-ascorbate shares the function of three paralogous proteins, UlaD/YiaQ, UlaE/YiaR and UlaF/YiaS, which encode a decarboxylase, a 3-epimerase and a 4-epimerase, respectively. In contrast, L-ascorbate enters the cells through the ula-encoded phosphotransferase transport system, but it is not carried by the yiaMNO-encoded ABC transporter. Proteomic analysis also indicated enhanced expression of the alkyl hydroperoxide reductase encoded by the ahpC gene, suggesting a response to oxidative stress generated during the aerobic metabolism of L-ascorbate. Control of ahpC expression by the OxyR global regulator in response to L-ascorbate concentration is consistent with the formation of hydrogen peroxide under our experimental conditions. The presence of certain amino acids such as proline, threonine or glutamine in the culture medium allowed aerobic L-ascorbate utilization by Escherichia coli cells. This effect could be explained by the ability of these amino acids to allow yiaK-S operon induction by L-ascorbate, thus increasing the metabolic flux of L-ascorbate dissimilation. Alternatively, these amino acids may slow the rate of L-ascorbate oxidation.

No abstract