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LL-diaminopimelate aminotransferase: the mechanism of substrate recognition and specificity

Amino acid biosynthesis is an essential process in living organisms. Certain amino acids can be synthesized by some organisms but not by others. L-Lysine is one of the essential amino acids that bacteria can synthesize but humans cannot. This is somewhat inconvenient for humans as much of their L-lysine must come from their diet. However, the lack of the lysine biosynthetic pathway in humans makes the bacterial enzymes within the pathway attractive drug targets. Recently, a novel lysine biosynthetic pathway was discovered in plants, Chlamydiae and some archaea. It is called the diaminopimelate aminotransferase (DAP-AT) pathway. In this pathway, LL-DAP-AT plays a key role by directly converting L-tetrahydrodipicolinate to LL-DAP in a single step. This is a quite interesting characteristic of LL-DAP-AT as the above conversion takes three sequential enzymatic steps in the previously known lysine biosynthetic pathways. Due to its absence in humans, LL-DAP-AT would be an attractive target for the development of novel antibiotics. In order to understand the catalytic mechanism and substrate recognition of LL-DAP-AT, the structural characterization of LL-DAP-AT is of paramount importance. In this thesis, the overall architecture of LL-DAP-AT and its substrate recognition mechanism revealed by the crystal structures of LL-DAP-AT from Arabidopsis thaliana and Chlamydia trachomatis will be discussed.
The crystal structure of the native LL-DAP-AT from A. thaliana (AtDAP-AT) presented in this thesis is the first structure of LL-DAP-AT to be determined. This structure revealed that LL-DAP-AT forms a functional homodimer and belongs to the type I fold family of PLP dependent aminotransferases. The subsequent determination of the substrate-bound AtDAP-AT structure showed how the two substrates, (LL-DAP and L-Glu) significantly different in size, are recognized by the same set of residues without significant conformational changes in the backbone structure. In addition, the LL-DAP-bound AtDAP-AT structure shows that the C-amino group of LL-DAP is recognized stereospecifically by the active site residues that are unique to the family of LL-DAP-AT enzymes.
Lastly, the chlamydial LL-DAP-AT presented in this thesis shows a new open conformation for LL-DAP-AT. The implications of the conformational flexibility of CtDAP-AT on the differences in substrate specificities among LL-DAP-AT are discussed.

Identiferoai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:AEU.10048/1649
Date06 1900
CreatorsWatanabe, Nobuhiko
ContributorsJames, Michael N. G. (Biochemistry), Glover, Mark J. N. (Biochemistry), Holmes, Charles F. B. (Biochemistry), Vederas, John C. (Chemistry), Anderson, Wayne F. (Molecular pharmacology and biological chemistry)
Source SetsLibrary and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada
LanguageEnglish
Detected LanguageEnglish
TypeThesis
Format59683557 bytes, application/pdf
RelationWatanabe, N., Cherney, M. M., van Belkum, M. J., Marcus, S. L., Flegel, M. D., Clay, M. D., Deyholos, M. K., Vederas, J. C., and James, M. N. G. (2007). Crystal structure of LL-diaminopimelate aminotransferase from Arabidopsis thaliana: a recently discovered enzyme in the biosynthesis of L-lysine by plants and Chlamydia. Journal of Molecular Biology. 371, 685-702., Watanabe, N., Clay, M. D., van Belkum, M. J., Cherney, M. M., Vederas, J. C., and James, M. N. G. (2008). Mechanism of substrate recognition and PLP-induced conformational changes in LL-diaminopimelate aminotransferase from Arabidopsis thaliana. Journal of Molecular Biology. 384, 1314-1329.

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