Fumarase, or fumarate hydratase, is a key metabolic enzyme. This enzyme catalyzes the reversible hydration of fumarate to L-malate in the seventh step of the Citric Acid Cycle. Mutations in the FH gene, which encodes for fumarase, lead to disease. Children that inherit two defective copies of FH develop a metabolic disease called fumarate hydratase deficiency, characterized by brain alterations, developmental delays, and early childhood mortality. In contrast, the inheritance of a single defective copy of FH is associated with Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC), which is associated with benign skin tumors and increased risk of kidney cancer. Here, we investigated three different clinically-relevant variants of fumarase that affect conserved residues near or in the active site: R233C, G282R, and Q376P. All three were previously identified in patients with FHD and/or HLRCC. The FH mutations were introduced by site-directed mutagenesis into a plasmid that encodes human fumarase using human codons. We purified wild-type and all three mutant fumarases using nickel affinity chromatography. We compared their enzymatic activities using fumarase assays and their oligomerizations states using Blue Native PAGE. We found that greater enzymatic defects correlated with worse clinical phenotypes. In addition, we unexpectedly found that all three variants were defective for oligomerization. Our research provides insight into how single missense mutations can lead to dramatic defects in protein structure and function, ultimately resulting in disease.

Biochemical analysis of three clinically-relevant variants of human fumarase

2019 Spring National Meeting

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