Oligomerization of wild‐type fumarase and a mutant associated with fumarate hydratase deficiency

Fumarase, or fumarate hydratase, is an enzyme that participates in the citric acid cycle and catalyzes the reversible hydration of fumarate to malate. Homozygous germline mutations in FH , the human fumarate hydratase gene, are linked to fumarate hydratase deficiency (FHD). Clinical features of this inborn error of metabolism include encephalitis, seizures, brain abnormalities, and developmental delay. Fumarase protein sequences are evolutionarily conserved, with human FH and Escherichia coli FumC sharing a stunning 59% amino acid sequence identity. Crystallographic analyses of E. coli and yeast fumarase have revealed that fumarase is homotetrameric, with two substrate binding sites, called the A‐ and B‐sites. Previously, the Weaver lab used X‐ray crystallography and kinetic experiments to investigate wild‐type FumC and FumC (E315Q), a clinically relevant mutant associated with FHD (Estevez, Skarda, Spencer, Banaszak, and Weaver, Protein Science 11:1552–7, 2002). They found that the E315 residue is conserved, lies within the A‐site, and is critical for catalysis. In this study, we purified both proteins and used fumarase assays and blue native PAGE (BN‐PAGE) to analyze their enzymatic activity and oligomerization states, respectively. Consistent with the findings from Estevez et al., we found that the E315Q mutant has greatly reduced enzymatic activity but is able to form tetramers. In future studies, we plan on analyzing other fumarase variants that have not been previously characterized. Support or Funding Information NSF