CRYSTALLOGRAPHIC STUDIES OF PROTEIN STRUCTURE/FUNCTION

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DESCRIPTION: The overall objective of this proposal is to understand the role of magnesium ion in the catalytic mechanisms of carbohydrate-processing metalloenzymes. Investigators also wish to learn how protein dynamics if coupled to metalloprotein function, and stability. To achieve these aims they will use novel methods that they have developed over previous budget periods, including cryocrystallography and time-resolved crystallography. There are three interdependent sub-projects within this overall project. The first aims to continue studies of xylose isomerase. Investigators will determine the structure of the initial E-S complex to deduce the role of the magnesium ions in catalysis of the ring-opening reaction. They will then simulate the entire catalytic pathway by combined quantum mechanics/molecular mechanics, to understand the unique catalytic properties of bridged bimetallic centers. The second project focuses on the role of magnesium in the reaction catalyzed by 3-isopropylmalate dehydrogenase. Investigators will use a combination of X-ray crystallography and genetic selection to map the correlations between thermal stability, protein flexibility, and metal-dependent activity in this essential amino-acid synthesizing enzyme. Finally, they will determine the structure and catalytic mechanism of the bifunctional mannose-6-phosphate isomerase from Pseudomonas aeruginosa. This enzyme catalyzes two non-contiguous reactions in alginate production: both reactions require magnesium ion. All three enzymes are amenable to low temperature or Laue crystallography, so that transient E-S complexes and reaction intermediates can be trapped and studied at high resolution, or are single substrate/single product enzymes, where the productive Michaelis complex can be observed directly in the crystal under equilibrium conditions.