The crystal structure of the bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase (DmpG)͞acylating acetaldehyde dehydrogenase (DmpF), which is involved in the bacterial degradation of toxic aromatic compounds, has been determined by multiwavelength anomalous dispersion (MAD) techniques and refined to 1.7-Å resolution. Structures of the two polypeptides represent a previously unrecognized subclass of metal-dependent aldolases, and of a CoA-dependent dehydrogenase. The structure reveals a mixed state of NAD ؉ binding to the DmpF protomer. Domain movements associated with cofactor binding in the DmpF protomer may be correlated with channeling and activity at the DmpG protomer. In the presence of NAD ؉ a 29-Å-long sequestered tunnel links the two active sites. Two barriers are visible along the tunnel and suggest control points for the movement of the reactive and volatile acetaldehyde intermediate between the two active sites. E nzymatic channeling is a process by which intermediates are moved directly between active sites in a sequential reaction pathway without equilibrating with the bulk phase (1, 2). Channeling processes are particularly advantageous over the free diffusion of reaction products through the bulk solvent because they can protect chemically labile intermediates from breakdown, prevent loss of nonpolar intermediates by diffusion across cell membranes, or protect the cell from toxic intermediates. Crystallographic studies on a number of different enzyme systems involved in substrate channeling (3-11) have revealed important structural factors that mediate intersubunit or interdomain communication and facilitate the efficient transfer of intermediates between distant active sites.A bifunctional aldolase-dehydrogenase catalyzes the final two steps of the meta-cleavage pathway for catechol, an intermediate in many bacterial species in the degradation of phenols, toluates, naphthalene, biphenyls and other aromatic compounds (reviewed in ref. 12). Thus, 4-hydroxy-2-ketovalerate aldolase (DmpG; EC 4.1.3.-) and acetaldehyde dehydrogenase (acylating) (DmpF; EC 1.2.1.10) from a methylphenol-degrading pseudomonad convert 4-hydroxy-2-ketovalerate to pyruvate and acetyl-CoA by way of the intermediate acetaldehyde (Scheme 1).The two enzymes are tightly associated with each other (13,14). Whereas the aldolase appears to be inactive when expressed without the dehydrogenase, the dehydrogenase retains some activity when expressed in the absence of aldolase (14), suggesting that at least the dehydrogenase active site is distinct from that of the aldolase. Several lines of evidence are consistent with channeling of acetaldehyde between the active sites. For example, the conversion of 4-hydroxy-2-ketovalerate to acetyl-CoA occurs Ϸ20 times faster than that of acetaldehyde to acetyl-CoA, and the K m for acetaldehyde exceeds 50 mM, a physiologically irrelevant concentration (J.P., unpublished data). In addition, it has been shown that the aldolase activity is stimulated by the addition of the dehydrogenase cofactor to t...

A high-throughput method for measuring transition metal content based on quantitation of X-ray fluorescence signals was used to analyze 654 proteins selected as targets by the New York Structural GenomiX Research Consortium. Over 10% showed the presence of transition metal atoms in stoichiometric amounts; these totals as well as the abundance distribution are similar to those of the Protein Data Bank. Bioinformatics analysis of the identified metalloproteins in most cases supported the metalloprotein annotation; identification of the conserved metal binding motif was also shown to be useful in verifying structural models of the proteins. Metalloproteomics provides a rapid structural and functional annotation for these sequences and is shown to be approximately 95% accurate in predicting the presence or absence of stoichiometric metal content. The project's goal is to assay at least 1 member from each Pfam family; approximately 500 Pfam families have been characterized with respect to transition metal content so far.

The three-dimensional crystallographic structure of the ybeY protein from Escherichia coli (SwissProt entry P77385) is reported at 2.7 A resolution. YbeY is a hypothetical protein that belongs to the UPF0054 family. The structure reveals that the protein binds a metal ion in a tetrahedral geometry. Three coordination sites are provided by histidine residues, while the fourth might be a water molecule that is not seen in the diffraction map because of its relatively low resolution. X-ray fluorescence analysis of the purified protein suggests that the metal is a nickel ion. The structure of ybeY and its sequence similarity to a number of predicted metal-dependent hydrolases provides a functional assignment for this protein family. The figures and tables of this paper were prepared using semi-automated tools, termed the Autopublish server, developed by the New York Structural GenomiX Research Consortium, with the goal of facilitating the rapid publication of crystallographic structures that emanate from worldwide Structural Genomics efforts, including the NIH-funded Protein Structure Initiative.