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Background:Fumarase, a significant enzyme of energy metabolism, catalyzes the reversible hydration of fumarate to L-malate. Mutations in the FH gene, encoding human fumarase, are associated with fumarate hydratase deficiency (FHD) and hereditary leiomyomatosis and renal cell cancer (HLRCC). Fumarase assembles into a homotetramer, with four active sites. Interestingly, residues from three of the four subunits within the homotetramer comprise each active site. Hence, any mutation affecting oligomerization is predicted to disrupt enzyme activity.Methods:We constructed two variants of hexahistidine-tagged human recombinant fumarase, A308T and H318Y, associated with FHD and HLRCC, respectively. Both Ala308 and His318 lie within the fumarase intersubunit interface. We purified unmodified human fumarase and the two variants, and analyzed their enzymatic activities and oligomerization states in vitro.Results:Both variants showed severely diminished fumarase activity. Steady-state kinetic analysis demonstrated that the variants were largely defective due to decreased turnover rate, while displaying Km values for L-malate similar to unmodified human recombinant fumarase. Blue native polyacrylamide gel electrophoresis and gel filtration experiments revealed that each variant had an altered oligomerization state, largely forming homodimers rather than homotetramers.Conclusion:We conclude that A308T and H318Y render human fumarase enzymatically inactive via defective oligomerization. Therefore, some forms of FHD and HLRCC can be linked to improperly folded quaternary structure.

Conjugation is the transfer of DNA from a donor to a recipient bacterium through a DNA translocation channel known as a Type IV Secretion System (T4SS). Our work focuses on the T4SS of ICEBs1, the integrative and conjugative element found in Bacillus subtilis. ConE is a component of the ICEBs1 T4SS. ConE is a VirB4‐like ATPase, most likely utilizing the energy generated from ATP hydrolysis for DNA transfer or T4SS assembly. Previously, we fused a hexa‐histidine tag (His6) to ConE for ease of purification via nickel affinity chromatography. However, we found that surprisingly low concentrations of imidazole inadvertently wash His6‐ConE off the nickel resin, preventing us from obtaining a high yield of pure protein. Here, we cloned deca‐histidine tagged ConE (His10‐ConE) and purified it side by side with His6‐ConE. We observed that His10‐ConE can be purified in higher yield and with fewer contaminants than His6‐ConE. After optimization, we conducted a large‐scale purification of His10‐ConE for the purpose of obtaining antigen for production of antibodies to ConE. In the future, the antibodies will be used in western blots, pull‐down assays, and immunofluorescence microscopy to characterize ConE levels, interactions, and localization, respectively. Additionally, we plan on exploring His10‐ConE's ability to interact with DNA using electrophoretic mobility shift assays.

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 InformationNSF