Aspartate-beta-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the biosynthetic pathway through which bacteria, fungi, and the higher plants synthesize amino acids, including lysine and methionine and the cell wall component diaminopimelate from aspartate. Blocks in this biosynthetic pathway, which is absent in mammals, are lethal, and inhibitors of ASADH may therefore serve as useful antibacterial, fungicidal, or herbicidal agents. We have determined the structure of ASADH from Escherichia coli by crystallography in the presence of its coenzyme and a substrate analogue that acts as a covalent inhibitor. This structure is comparable to that of the covalent intermediate that forms during the reaction catalyzed by ASADH. The key catalytic residues are confirmed as cysteine 135, which is covalently linked to the intermediate during the reaction, and histidine 274, which acts as an acid/base catalyst. The substrate and coenzyme binding residues are also identified, and these active site residues are conserved throughout all of the ASADH sequences. Comparison of the previously determined apo-enzyme structure [Hadfield et al. J. Mol. Biol. (1999) 289, 991-1002] and the complex presented here reveals a conformational change that occurs on binding of NADP that creates a binding site for the amino acid substrate. These results provide a structural explanation for the preferred order of substrate binding that is observed kinetically.
Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure with significant predisposition to the development of poor prognosis myelodysplasia and leukemia, exocrine pancreatic failure and metaphyseal chondrodysplasia. Although the SBDS gene mutated in this disorder is highly conserved in Archaea and all eukaryotes, the function is unknown. To interpret the molecular consequences of SDS-associated mutations, we have solved the crystal structure of the Archaeoglobus fulgidus SBDS protein orthologue at a resolution of 1.9 Å, revealing a three domain architecture. The N-terminal (FYSH) domain is the most frequent target for disease mutations and contains a novel mixed ␣/-fold identical to the single domain yeast protein Yhr087wp that is implicated in RNA metabolism. The central domain consists of a three-helical bundle, whereas the C-terminal domain has a ferredoxin-like fold. By genetic complementation analysis of the essential Saccharomyces cerevisiae SBDS orthologue YLR022C, we demonstrate an essential role in vivo for the FYSH domain and the central three-helical bundle. We further show that the common SDS-related K62X truncation is non-functional. Most SDS-related missense mutations that alter surface epitopes do not impair YLR022C function, but mutations affecting residues buried in the hydrophobic core of the FYSH domain severely impair or abrogate complementation. These data are consistent with absence of homozygosity for the common K62X truncation mutation in individuals with SDS, indicating that the SDS disease phenotype is a consequence of expression of hypomorphic SBDS alleles and that complete loss of SBDS function is likely to be lethal.
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