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Structure and Regulation of Aspartate Pathway Enzymes and Deuteration Effects on Protein Structure

by Liu, Xuying

Abstract (Summary)
The aspartate biosynthetic pathway, essential in plants and in most bacteria and fungi, can produce the amino acids threonine, lysine, methionine, and isoleucine. The first commitment step of this pathway is catalyzed by aspartokinase (AK). The archeal thermophilic Methanococcus jannaschii has only a single, monofunctional form of AK. X-ray crystallographic studies indicate that mjAK is a tetramer in the crystalline state, which coincides with the oligomeric structure in solution. The substrate L-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of E. coli AK.5 Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. The allosteric inhibitor, L-threonine, cooperatively binds to two sites in each monomer of this dimer of dimers, one site in the regulatory domain interface and the other in the kinase domain close to the proposed L-aspartate binding site. The primary L-threonine binding in the regulatory domain interface induces a regulatory domain movement, leading to an open structure and fixes the enzyme in this inhibited conformation. The secondary L-threonine binding in the kinase domain causes a critical substrate residue Arg207 movement, resulting in an unfavorable L-asparate binding pocket. The second commitment step of aspartate biosynthetic pathway is catalyzed by aspartate-?-semialdehyde dehydrogenase (ASADH). As a potential novel antifungal drug target, ASADH from Candida albicans (caASADH) was cloned for overproduction of the enzyme, subsequentially purified and screened for crystallization. The resulting caASADH crystals diffracted to modest resolution, but molecular replacement has not yielded a useful solution. Our comprehensive understanding of the catalytic mechanism of aspartate-?-semialdehyde dehydrogenase allows further inhibitor development to target this essential enzyme from infectious microorganisms. Fragment-based lead discovery is being evaluated as a rapid approach to identify inhibitory fragments. Unexpectedly, ASADHs from Gram-positive and Gram-negative species show different preferences against the a fragment library, even though their active sites are highly conserved. This specificity suggests that highly selective antibiotics can be developed to target either Gram-negative or Gram-positive infectious bacteria. As the first step towards structure determination with neutron diffraction, deuteration effects were investigated for haloalkane dehalogenase from Xanthobacter autotrophicus (XaDHL). The enzyme was overexpressed under different isotopic conditions to produce fully hydrogenous (h-XaDHL) and perdeuterated (d-XaDHL) enzyme forms. Optimal crystals of h-XaDHL and d-XaDHL were obtained at different pH conditions with similar P21212 unit cells. X-ray diffraction data on these crystals were refined to high resolution with excellent overall statistics. The overall conformations of h-XaDHL and d-XaDHL are nearly identical. One significant difference is that h-XaDHL was found to have a more hydrophobic core than d-XaDHL. Unexpectedly, Asp124, the primary nucleophile, is displaced from its position in h-XaDHL and rotates to form a hydrogen bond with His289 in d-XaDHL, excluding the second nucleophile water molecule from the active site. This unusual active site configuration represents the proposed termination state of the catalytic reaction and provides an explanation for the acid inhibition of XaDHL. In summary, structural studies of threonine-sensitive AK have provided new insights into the regulation mechanism of the aspartate pathway. Structural studies of ASADHs from yeast and fungal organisms will extend this target for antifungal drug development, while our ASADH kinetics assay was shown to be an efficient approach for fragment-based lead screening to target this essential enzyme. Our observed perdeuteration effects on protein structure underline the importance of carefully verifying the assumption that isotopic substitution does not produce structural changes in protein structures.
Bibliographical Information:

Advisor:

School:University of Toledo

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:aspartokinase allosteric inhibition threonine sensitive aspartate semialdehyde dehydrogenase deuteration effects

ISBN:

Date of Publication:01/01/2008

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