The overall objective of this research program 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, we use novel methods that we have developed over the last several years, including cryocrystallography and time-resolved crystallography. There are three interdependent sub-projects within this overall project. The first continues studies of xylose isomerase. The investigators are determining the structure of the initial E-S complex to deduce therole of the magnesium ions in catalysis of the ring-opening reaction. We 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 usea combination of X-ray crystallography and genetic selection to map the correlations between thermal stability, protein flexibility, and metal-dependent activity in this essential arnino-acid synthesizing enzyme. Finally, we will determine the structure and catalytic mechanism of the bifunctional mannose-6-phosphate isomerase from Pseudomonay 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-Scomplexes and reaction intermediates can be trapped and studied at high resolution; or are single substrate/single product enzymes, where the productive Nfichaelis complex can be observed directly in the crystal under equilibrium conditions.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR010888-05
Application #
6345236
Study Section
Project Start
2000-07-01
Project End
2002-06-30
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
5
Fiscal Year
2000
Total Cost
$4,940
Indirect Cost
Name
Boston University
Department
Type
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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