Fumarase (EC 4.2.1.2), or fumarate hydratase (FH), catalyzes the reversible hydration of fumarate to L-malate [1, 2]. Fumarase enzymes are found in all three domains of life and are strikingly conserved. For example, human and Escherichia coli fumarase share 60% amino acid sequence identity [3] and human fumarase can complement a fum1 deletion in yeast [4]. Moreover, the (GS³⁶⁵SxxPxK³⁷¹xN³⁷³) signature sequence can be used to identify fumarase family members [5]. All amino acid numbering within this manuscript is based upon the human fumarase protein sequence, P07954 [6]. The human fumarate hydratase gene (FH), located on chromosome 1q42.1 [7], encodes both mitochondrial and cytosolic isoforms of fumarase [8]. Mitochondrial fumarase participates in the Krebs cycle, whereas cytosolic fumarase assists in the metabolism of fumarate, a by-product of amino acid metabolism and the urea cycle [9, 10].

Mutation of FH is associated with a rare metabolic disease known as fumarate hydratase deficiency (FHD) or fumaric aciduria (OMIM 606812; reviewed in [11-14]). FHD is inherited in an autosomal recessive manner with individuals exhibiting low levels of fumarase activity in fibroblasts or other cells, and high levels of fumaric acid in urine. Some symptoms of FHD include hypotonia, cerebral malformation and atrophy, seizures, failure to thrive, and developmental delay [15-18]. As therapeutic strategies are lacking, many individuals with FHD do not survive past early childhood.

Fumarase also functions as a tumor suppressor, where certain germline loss-of-function mutations in FH predispose individuals to a variety of tumors and cancers [19-24]. Mutations in FH are closely associated with multiple cutaneous and uterine leiomyomatosis syndrome (MCUL), manifested by the development of benign smooth tumors in the skin and uterus. This disease is now referred to as hereditary leiomyomatosis and renal cell cancer (HLRCC; OMIM 150800), due to more recent recognition of the associated increased risk for kidney cancer. More recently, FH mutations have been linked to paragangliomas and pheochromocytomas, tumors in the neuroendocrine tissues and the adrenal medulla, respectively [25, 26] as well as bladder, breast [27], and testicular cancers [28]. Several mechanisms have been proposed to explain how FH functions as a tumor suppressor [4, 29-37].

More than 100 mutations in the FH gene associated with disease have been reported so far, with the majority being missense mutations [38]. Most clinically relevant missense mutations affect residues that are evolutionarily conserved [39, 40]. Other mutations, such as deletions or splice-site mutations, often result in omission of whole segments of the protein and thus cause severe disruptions in conformation of the protein [25, 41, 42]. Overall, mutations in FH seem to cause structural changes to the enzyme that eliminate or compromise enzymatic function. Many efforts have been made to link particular mutations to the structure and function of fumarase. The crystal structures of E. coli fumarase C (FumC) [43], yeast fumarase [44], and human fumarase [40] are highly similar, consistent with their high sequence identity. Fumarase is a homotetramer, with each fumarase subunit constructed from three domains termed D1, D2, and D3 (PDB ID: 3E04 [40]; Fig. (1A). The central domain, D2, facilitates tetramerization (Fig. 1B). Each of the four independent active sites, deduced by co-crystals with inhibitors and through biochemical analyses of point mutants [43, 45-47], are located between domains D1 and D3 at the four corners of the enzyme (Fig. 1B).

Structurally, each human fumarase subunit harbors twenty-three α-helices and eight β-strands (Fig. 2A) [40]. Amino acid alignments conducted on fumarase and other superfamily members, including aspartase, arginosuccinate lyase, adenylosuccinate lyase, and carboxy cis-cis muconate lactonizing enzyme, have identified three regions of significant conservation (Fig. 2A) [5]. From a three-dimensional perspective, these three conserved areas lie in disparate areas of the monomeric structure (Fig. 2B). However, upon tetramerization these regions coalesce to form the four active sites. More specifically, residues 176-193, 228-247 and 359-381, donated from three distinct subunits, form each fumarase active site. The first and third conserved regions construct the majority of the substrate-binding site, while the second and third regions donate the catalytic groups H235 (catalytic acid) and S365 (catalytic base), respectively (Fig. 2B) [1].

More than 50 FH mutations have been mapped onto the crystal structure of fumarase [39-41, 48]. Many mutations are located in or near the active site, and thus likely affect enzymatic activity directly. A few mutations affecting the active site of E. coli FumC have been shown to decrease activity of the purified enzyme [3, 45, 46]. In principle, a mutation that affects oligomerization should affect enzymatic activity as each of the four active sites in the enzyme is composed of residues donated from three of the four subunits of the homotetramer (Figs. 2A and 2B) [46]. A few reports have suggested that fumarase is only active as a tetramer [49, 50]. The cited evidence pertains to experiments that showed that fumarase treated with guanidine hydrochloride, urea, or organic solvents becomes both monomeric and enzymatically inactive. However, these reagents would also denature the protein, which could also explain the observed loss of activity.

Fig. (1). Tertiary and quaternary structure of human fumarase (PDB ID: 3E04 [40]). (A) A single subunit of human fumarase has been colored-coded by domain: domain 1 (D1) – red, domain 2 (D2) – yellow, and domain 3 (D3) – blue. (B) The human fumarase homotetramer has been colored by chain, with subunits labeled as A, B, C, or D. The all α-helical central domain, D2, forms the major intersubunit interface upon oligomerization. D1 and D3 domains lie at the corners of the homotetramer and outline the entry point to the four independent fumarase active sites.

Another analysis of a very common HLRCC FH variant (R190H) suggests that tetramerization is required for activity [51]. Over-expression of FH (R190H) in cells containing wild-type FH causes a dominant-negative loss of fumarase activity and loss of FH tetramers. R190 forms an ion pair with a glutamic acid residue from the same subunit and is located on the section that contributes the active site histidine. Two more interesting structural notes follow: (1) R190 also forms a hydrogen bond with the carbonyl oxygen of a serine residue from a neighboring subunit and (2) the hydrogen bond shared between R190 and the glutamic acid is located within a hydrophobic region (residues donated from 3 of 4 subunits). The hydrophobic region would serve to strengthen the hydrogen bond shared between these two charged residues. So, it is not surprising that the R190H would be deleterious to fumarase structure and function.

In this study, we purified and characterized the enzymatic activities and oligomerization states of recombinant human fumarase (HsFH) and two disease variants of the enzyme (A308T and H318Y). The A308T mutation (c.922G>A in exon 7) was previously identified in a patient with FHD that presented with hypotonia, microcephaly, and slow development [17, 52]. The other mutation investigated, H318Y (c.952A>T in exon 7), has been found in several patients diagnosed with HLRCC or MCUL [53-55]. Both alanine 308 and histidine 318 are located in the central D2 domain (Figs. 2A and B) [43] and were previously hypothesized to disrupt intersubunit interactions based on their locations in the crystal structure of human fumarase (PDB ID: 3E04) [40]. Our biochemical analyses of the variants indicate that both are defective for enzymatic activity and oligomerization. Our data provide new biochemical insights into how mutations in FH lead to disease and provide support for the model that a functional active site requires tetramer formation. Throughout the manuscript, we refer to unmodified recombinant human fumarase as HsFH, while the two variants are identified as A308T and H318Y, respectively.

Fig. (2). Primary and tertiary structure representations of the fumarase family highly conserved regions. (A) The primary sequence of human fumarase (P07954: FUMH_HUMAN) has been annotated with secondary structural elements, fumarase family highly conserved regions, active site residues H235 and S365, and the clinically observed variant (A308 and H318) locations. The three regions of high conservation observed within the fumarase family have been colored accordingly: cyan (region 1 residues 176-193), dark green (region 2 residues 228-247), and magenta (region 3 residues 359-381). Region 3 also defines the “signature sequence” for the fumarase super-family. Regions 1 and 3 form the majority of the substrate-binding site, while regions 2 and 3 contribute the catalytic groups H235 and S365 (denoted by the double dagger symbol). Additionally, A308 and H318 have been denoted with an asterisk and colored red and blue, respectively. (B) The tertiary structure of the fumarase monomer (PDB ID: 3E04 [40]) emphasizes the three-dimensional juxtaposition of the highly conserved regions within the fumarase family. The tertiary structure has been colored and annotated similar to panel (A). The three-dimensional view of the fumarase monomer emphasizes (1) the spatial distance between the highly conserved regions (1-3) and (2) the locations of A308 and H318 near α-helices 12 and 13 of D2.

Given our gel filtration and BN-PAGE results, we propose that both point mutants prevent stable HsFH tetramerization, and thereby disrupt formation of the active site leading to diminished catalytic function. Fumarase catalytic activity is inherently tied to homotetramer formation, as residues from three different subunits are required to assemble a functional active site [1, 43]. Structurally, A308 and H318 lie within regions forming intersubunit interactions, including α-helices 12 - 14 (Fig. 7A). More specifically, A308 lies buried within a loop connecting α-helices 11 and 12. The buried region around A308 also includes A273 and A314 as donated by the loop between α-helix 10 and 11, and α-helix 12, respectively (Fig. 7B). Therefore, the local region surrounding A308 is structurally restricted and incompatible with bulky side chain placement. Thus, threonine substitution at this location (A308T) would certainly require conformational adaptation to accommodate this β-branched and polar side chain. The gel filtration and BN-PAGE results support structural adaptation within the A308T variant via homodimer formation (Fig. 6A).

The localization of H318, a polar/charged side chain, within the solvent inaccessible core of human fumarase is atypical. This atypical localization of H318 within the solvent inaccessible region of HsFH is electrostatically accommodated via an intrasubunit hydrogen bond with K409 (Fig. 7B). Interestingly, D319 (one residue removed from H318 within α-helix 13) is also buried at the solvent inaccessible fumarase subunit interface. Alternatively, D319 is located at an intersubunit interface. D319 (subunit A) forms a hydrogen bond with Q254 (subunit D) to stabilize this dimer interface (Fig. 7B). Thus, the intrasubunit hydrogen bond between H318 and K409 facilitates formation of the intersubunit hydrogen bond shared between Q254 and D319 as donated by α-helices 10 and 13 from subunits D and A, respectively. Structurally, the region occupied by H318 would be unable to accommodate the substitution of the larger tyrosine side chain. Ultimately, the replacement of histidine 318 with a tyrosine (H318Y) may impose structural consequences that disrupt both the H318:K409 intrasubunit and D319:Q254 intersubunit electrostatic interactions leading to destabilization of the 3,000 Ų A-D intersubunit interface (Fig. 3A). The gel filtration and BN-PAGE results for H318Y (Fig. 6A) support this model.

Fig. (7). The putative structural impact of the A308T and H318Y variants at the multi-subunit active site in the human fumarase structure (PDB ID: 3E04 [40]). (A) The human fumarase tetramerization interface has been displayed looking down related D2 domains. For clarity, D1 and D3 have been removed from this image. Upon oligomerization, α-helix 14 lies at the center of the homotetramer. Additionally, α-helices 12 and 13 contribute residues to the tetramer interface. (B) The three-subunit fumarase active site has been depicted with emphasis upon the locations of A308 and H318 in context to the A-D dimer interface and fumarase signature sequence (GS365SxxPxK371xN373). Dashed lines depict hydrogen bonds and the dot surface reflects the A308 Cβ atom van der Waal radius. Residual monomer elements have been colored as depicted in Fig. (1B). The side chain atoms for A308 and H318 have been depicted in stick format and colored by atom type (red-oxygen, blue-nitrogen, orange-carbon). The fumarase active site water (wat) has been depicted as a magenta colored sphere.

How do the oligomerization defects fit with our kinetic results where A308T and H318Y have K_m values similar to HsFH yet diminished turnover numbers? Past investigations have suggested that regions from three of the four subunits are required for a functional fumarase active site [43, 47]. Therefore, we hypothesize that the residual A308T and H318Y activity (Table 1) is due to the observed small fraction of homotetrameric species (Fig. 6A). While the turnover rates of A308T and H318Y are >900-fold reduced compared to HsFH, their defects in tetramerization do not seem to be as profound. Thus, it is likely that the atomic-level alterations of the residue substitutions on protein structure, such as the disruption of significant electrostatic interactions described in the prior paragraph, have additional negative consequences on active site formation. In other words, although a small fraction of the mutant proteins can form tetramers (Fig. 6A), the tetramers are not enzymatically active due to small structural changes that cannot be detected with gel filtration or BN-PAGE. Additional higher resolution structural studies are required to detect these subtle differences in structure.

We hypothesize the C-D dimer (Fig. 3B) as the putative species observed during our gel filtration and BN-PAGE studies. We will use the amount of surface area in conjunction with the proximity of A308 and H318 to establish support for the C-D dimer as the experimentally observed dimer. According to PISA [62], the three dimer combinations ranked in order of decreasing buried surface area (Ų) are A-D, C-D, and B-D (Fig. 3). Thus, the greatest amount of shared interface resides in the A-D dimer, followed by the C-D dimer and lastly the B-D dimer. The calculated A-D dimer interface is the most stable. However, the arrangement of the paired A308 and H318 side chains within each subunit align closest within the A-D dimer (Figs. 3A and 7B). Likewise, alteration of these residues would have the most impact on α-helices 12 and 13 in the A-D dimer. The B-D dimer has very little shared surface area and an average ΔG (P-value) of 0.522. A ΔG (P-value) of this magnitude suggests limited interaction specificity. Therefore, we hypothesize the C-D dimer as the species observed during our gel filtration and BN-PAGE studies. The C-D dimer has a related average ΔG (P-value) of 0.057, which indicates high specificity for this dimer interface [62]. Interestingly, the C-D dimer (symmetrical to the A-B dimer illustrated in (Fig. 7B) contributes the two highly conserved regions (Fig. 2) that build the substrate-binding site.

In conclusion, our results indicate that recombinant human fumarase purified from E. coli is enzymatically active. Furthermore, we have shown that the A308T and H318Y variants are both enzymatically inactive and defective for oligomerization. Therefore, some forms of FHD and HLRCC may be linked to improperly folded quaternary structure.