Mandelamide Hydrolase: Structural Studies of a Novel Amidase
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چکیده
Mandelamide Hydrolase: Structural Studies of a Novel Amidase A thesis presented to the Biochemistry Department Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Matthew Hope Mandelamide hydrolase(MAH), the first enzyme in the mandelamide pathway, catalyzes the conversion of mandelamide in Psuedomonas putida which allows the species to grow without gluclose. MAH has not been structurally characterized to atomic resolution yet, and doing so would yield valuable information for studying the ways in which simple species evolve new functions through mutagenesis. In this study, we explored three methods in stabilizing MAH to in order to better facilitate crystallization and obtain a 3D-structure. Constructs of the enzyme were rationally designed from a homology model to test the effects of foreshortening the amino acid sequence. Also, dynamic light scattering (DLS) experiments were employed to find conditions that will monodisperse laser light, indicating that the protein is more likely to crystallize than aggregate. Finally, thermal denaturation curves were performed under various conditions to find ways to thermally stabilize the enzyme in order to minimize unfolding and aggregation and promote crystallization. Although this inquiry did not yield crystals of MAH, DLS conditions containing dithiothreitol (DTT) were found to be beneficial for minimizing polydispersion and the thermal denaturation curves indicated that 6-dimethyl-4-heptyl-β-Dmaltoside improves the thermal stability. Qualitatively, foreshortening the protein also improves the quality of crystallization drops. These results can be utilized in the future to stabilize MAH and elucidate the proper crystallization conditions that could eventually lead to a structure at atomic resolution.
منابع مشابه
Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633.
The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarb...
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