Abstract
Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes a critical branch point transformation in amino acid bio-synthesis. The products of the aspartate pathway are essential in microorganisms, and this entire pathway is absent in mammals, making this enzyme an attractive target for antibiotic development. The first structure of an ASADH from a Gram-positive bacterium, Streptococcus pneumoniae, has now been determined. The overall structure of the apoenzyme has a similar fold to those of the Gram-negative and archaeal ASADHs but contains some interesting structural variations that can be exploited for inhibitor design. Binding of the coenzyme NADP, as well as a truncated nucleotide analogue, into an alternative conformation from that observed in Gram-negative ASADHs causes an enzyme domain closure that precedes catalysis. The covalent acyl-enzyme intermediate was trapped by soaking the substrate into crystals of the coenzyme complex, and the structure of this elusive intermediate provides detailed insights into the catalytic mechanism.
Highlights
Tional product of this pathway, S-adenosylmethionine, is an essential methyl group donor that can serve, after decarboxylation, as a propylamine donor in the synthesis of the polyamines spermidine and spermine
aspartate--semialdehyde dehydrogenase (ASADH) catalyzes the production of aspartate--semialdehyde, the first branch point intermediate in the aspartate pathway, and the structure of the enzyme has been determined from several infectious Gram-negative bacteria (10 –12)
Overall Structure of S. pneumoniae ASADH—The structure of apo spASADH was solved by MR using a combined ensemble of ASADH search models, each with less than 30% sequence identity to spASADH
Summary
Enzyme Expression and Purification—The asd gene encoding ASADH was amplified from a S. pneumoniae genomic DNA template purchased from American Type Culture Collection (ATCC). Crystallization and Data Collection—Initial crystallization conditions were identified by sparse matrix screening of spASADH at a protein concentration of 10 mg/ml Both index screens (Hampton Research) and JBScreen Basic (Jena Bioscience) yielded initial crystals for the apoenzyme. Homology models of several ASADHs (Protein Data Bank codes 1GL3, 1NWH, 1MB4, and 1YS4) were superimposed, and the nonhomologous regions were trimmed to generate an ensemble of ASADH search models containing only the conserved regions This ensemble was used to find the correct orientation of spASADH, because the use of any individual ASADH model had failed to yield a solution. The model of mjASADH (Protein Data Bank code 1YS4) with the highest sequence identity to spASADH was subjected to rigid body refinement followed by simulated annealing refinement in CNS [16] to obtain the initial electron density map. Surface area calculations were performed using Surface Racer 4.0 [21] with a solvent probe radius of 1.4 Å
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