With over 140 vitamin B6 (Pyridoxal 5’-phosphate, PLP) dependent enzymes, serving vital roles in various transamination, decarboxylation, retro-aldol cleavage and synthesis pathways these enzymes constitute the most versatile catalytic systems in nature. Enzymes of this group have an inherent reaction as well as substrate specificity. A single co-factor namely, PLP is used by specific enzymes of this group to serve distinct roles during the catalytic reaction. An ordered evolutionary adaptation in these enzymes has led to specialization achieved by each enzyme for catalyzing specific reactions. L-Threonine aldolase (L-TA) is one such PLP- dependent enzyme that catalyzes the retro-aldol cleavage of several β-hydroxy amino acids, although its natural substrates are L-threonine and L-allo-threonine with the enzyme having significant preference for L-allo-threonine. It also catalyzes racemization and transamination of D-alanine but not of the β-hydroxy amino acids. Thus, the enzyme exhibits both substrate and reaction specificity. Although, L-TA is frequently employed for stereoselective synthesis of pharmaceutically useful compounds, its reaction mechanism and associated specificity is still not clearly understood. L-TA from Escherichia coli (eTA) is being studied in our laboratory. Our objective is to elucidate the catalytic mechanism of eTA and its mode of substrate and reaction specificity using X-ray crystallography. Another objective is to establish evolutionary relationship of L-TA with other B6-dependent enzymes, such as serine hydroxymethyltransferase (SHMT) and Thermatoga maritima L-TA (TTA) that have the same fold and catalyze similar reactions. Our structural studies show that while the crystal structures of the two L-TAs are similar, they are significantly different from that of SHMT, especially at the active site. In the L-TA structures, a loop with proposed important active site residue, His126 is replaced by tetrahydrofolate (THF) in SHMT. The crystal structures of eTA in its native form and in complex with substrate or product have highlighted the importance of His126 in ensuring substrate specificity during retro-aldol cleavage of various β-hydroxy amino acids and His83 or a conserved water molecule to be active site base. Our study emphasizes the molecular level implications of the catalytic mechanism of eTA.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-3261 |
| Date | 23 July 2010 |
| Creators | Govinda, Remsh Soumya |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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