Molecular Evolution of Pyridoxal Phoshate Enzymes

University Of California Berkeley, Berkeley CA

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Abstract

DESCRIPTION (provided by applicant): The major advances in understanding protein design and function arising from genomics, and computational and structural biology, have enabled lines of enquiry to enzymology, that could not have been undertaken until recently. Directed evolution or DNA shuffling will be used to attempt to convert an important oxidation enzyme of intermediary metabolism, malate dehydrogenase, to acquire the activity of a structurally related enzyme, lactate dehydrogenase, in order to compare the results of laboratory with natural evolution. The same technology will be employed, additionally, to attempt to narrow the specificity of tyrosine aminotransferase, an enzyme that reacts with aromatic and negatively charged amino acids, to an activity that engages only the latter class. Directed evolution of a plant enzyme, involved in the production of the gaseous hormone, ethylene, will be used to attempt to convert it into an aminotransferase, a very distantly related enzyme. Human tyrosinemia type 2, has been shown to be caused by a few mutations. These mutant enzymes will be characterized, both with respect to their catalytic properties and their stability under near physiological conditions, in order to define the precise relation of the molecular defect to the disease. It is widely appreciated, that a large fraction of genome sequences (perhaps 40%) has been misannotated. A test set of diverse genome-annotated aspartate and tyrosine aminotransferases, will be prepared and characterized, for substrate specificity, in order to try to discover better rules for genome annotation of enzymes. There are very few drugs that work by disrupting protein/protein interaction. A major reason is that assays are cumbersome and expensive. A mass spectrometric method is being devised, which allows rapid high throughput assays for this purpose.