Crystal structures of native and recombinant yeast fumarase

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Fumarase catalyzes the reversible hydration/dehydration of fumarate to malate.
• Understanding fumarase structure is crucial for metabolic pathway analysis.

Purpose of the Study:

• Determine the crystal structures of native and recombinant yeast fumarase.
• Investigate the active site and catalytic mechanism of fumarase.

Main Methods:

• X-ray crystallography of native and recombinant Saccharomyces cerevisiae fumarase.
• Expression of recombinant fumarase in Escherichia coli.
• Crystallization with and without inhibitors (meso-tartrate).

Main Results:

• Moderate resolution crystal structures of both native (NY-fumarase) and recombinant (RY-fumarase) yeast fumarase were obtained.
• Both structures revealed a homo-tetrameric protein with three domains per subunit, lacking the N-terminal translocation segment.
• The active site is formed by residues from three polypeptide chains; the recombinant form showed an unoccupied active site, while the native form bound meso-tartrate.
• Meso-tartrate binding site is near the E. coli fumarase citrate-binding site and involves a water molecule.

Conclusions:

• The crystal structures provide insights into the three-dimensional structure of yeast fumarase.
• The role of an active-site water molecule in the fumarase reaction mechanism is supported by structural data.
• Independent determination of native and recombinant structures validates findings.