Aspartate semialdehyde dehydrogenase (ASADH) is an essential enzyme that functions in the biosynthesis of isoleucine, lysine, methionine, and threonine in bacteria. ASADH catalyzes the NADPH-dependent reduction of b-aspartyl phosphate to aspartate b-semialdehyde, while also forming inorganic phosphate (Pi) as a product. The importance of this enzyme in bacteria was highlighted in a study whereby each of the 4,101 genes of the Bacillus subtilis genome was individually inactivated. Results demonstrated that asd was one of only 271 genes found to be essential. The overall aim of this study is to validate that ASADH is an essential enzyme in Gram-positive and Gram-negative bacteria, with a view to facilitate the drug discovery progress.
The bacterium Methicillin-resistant Staphylococcus aureus (MRSA) is a significant human pathogen. It is responsible for diseases ranging from minor skin infections to more serious and life threatening conditions such as bacteremia, septic shock, pneumonia, meningitis, and endocarditis. The organism is of real concern because of its resistance to commonly used frontline antibiotics. We have cloned, expressed, and purified His-tagged ASADH from the Gram-positive pathogen MRSA. We show by circular dichroism spectroscopy that His-tagged MRSA-ASADH is folded and purified enzyme was determined to be active using an established assay. The quaternary structure of His-tagged MRSA-ASADH was examined by analytical ultracentrifugation. Sedimentation velocity experiments show that His-tagged MRSA-ASDH exists as two species in solution with sedimentation coefficients (s20,w) of 2.5 S and 4.0 S, and molecular masses of 33 kDa and 69 kDa, respectively. We have isolated genomic DNA of S. aureus Newman strain and amplified the asd gene with approximately 350bp upstream and downstream regions. The wild-type asd gene was partially deleted and was incorporated into the chromosome of S aureus Newman via homologous recombination. Future studies will examine the essentiality of ASADH in S. aureus.