The multisubunit active site of fumarase C from Escherichia coli

Weaver, Todd; Levitt, David G.; Donnelly, Mark I.; Stevens, P P; Banaszak, Leonard J.

doi:10.1038/nsb0895-654

Cited by 123 publications

(174 citation statements)

References 36 publications

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“…These correspond to region 1 H133 to T150, region 2 G189 to E204, and region 3 G321 to E335 in human FH. 9 These have been shown to be important in formation of the active A-site and B-site and have some overlap with the regions that may represent mutational hotspots (Figure 2). The clustal alignment across seven species showed that, of distinct missense mutations of FH in MCUL, 16 of 26 (60%) were of fully conserved, 5 of 26 (20%) of strongly conserved, 4 of 26 (16%) of weakly conserved, and 1 of 26 (4%) of nonconserved residues (Table 2, Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…The crystal structures of the E. coli fumarase C and Saccharomyces cerevisiae fumarase have been elucidated. 9,10 The high degree of homology of these Supported by Cancer Research UK (clinical research fellowship to N.A.A. ).…”

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confidence: 99%

“…The crystal structures of the E. coli fumarase C and Saccharomyces cerevisiae fumarase have been elucidated. 9,10 The high degree of homology of these proteins to human FH allows their use as models for predicting the effect of missense mutations on FH function. The FH protein exists as a homotetramer with two substrate-binding sites (Figure 1), designated the A-site and the B-site.…”

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confidence: 99%

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Missense Mutations in Fumarate Hydratase in Multiple Cutaneous and Uterine Leiomyomatosis and Renal Cell Cancer

Alam

Olpin

Rowan

et al. 2005

The Journal of Molecular Diagnostics

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Heterozygous germline mutations in fumarate hydratase (FH) predispose to the multiple cutaneous and uterine leiomyomatosis syndrome (MCUL), which, when co-existing with renal cancer, is also known as hereditary leiomyomatosis and renal cell cancer. Twenty-seven distinct missense mutations represent 68% of FH mutations reported in MCUL. Here we show that FH missense mutations significantly occurred in fully conserved residues and in residues functioning in the FH A-site, B-site, or subunit-interacting region. Of 24 distinct missense mutations, 13 (54%) occurred in the substrate-binding Asite, 4 (17%) in the substrate-binding B-site, and 7 (29%) in the subunit-interacting region. Clustering of missense mutations suggested the presence of possible mutational hotspots. FH functional assay of lymphoblastoid cell lines from 23 individuals with heterozygous FH missense mutations showed that A-site mutants had significantly less residual activity than B-site mutants, supporting data from Escherichia coli that the A-site is the main catalytic site. Missense FH mutations predisposing to renal cancer had no unusual features, and identical mutations were found in families without renal cancer, suggesting a role for genetic or environmental factors in renal cancer development In the autosomal dominant syndrome of multiple cutaneous and uterine leiomyomatosis (MCUL, Reed syndrome, leiomyomatosis cutis et uteri, multiple leiomyomatosis; OMIM 150800), affected females develop uterine leiomyomas and affected individuals of both sexes develop cutaneous leiomyomas. 1 Cutaneous leiomyomas are believed to be derived from the smooth muscle of the pilo-arrector apparatus. They generally present in the second, third, or fourth decades, typically as grouped papules or nodules on the trunk or limbs and are characteristically painful particularly in response to low temperatures or touch. Uterine leiomyomas or fibroids in MCUL are severely symptomatic with a large proportion of patients requiring symptom control by hysterectomy. 2 A small proportion of families with MCUL also cluster renal cancer, either papillary renal type II cancer or renal collecting duct cancer. 1,[3][4][5][6] This disease variant has been referred to as hereditary leiomyomatosis and renal cancer (HLRCC, OMIM 605839). MCUL/HLRCC has been found to be caused by germline mutations in fumarate hydratase (FH) in the majority of screened cases. 1,5,6 Forty-six distinct FH mutations have been reported to date in MCUL/HLRCC. Twenty-seven of these are missense mutations of 26 different residues (one residue has two reported mutations, R190H and R190L). These 27 distinct missense mutations represent 55 of 81 (68%) of the FH mutations reported in MCUL probands. 1,[5][6][7] The FH locus encodes two isoforms of fumarate hydratase, cytosolic and mitochondrial, which differ only in that the latter has an initial mitochondrial signal peptide. Fumarate hydratase catalyzes the stereospecific reversible hydration of fumarate to L-malate. The mitochondrial isoform performs this reactio...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Missense Mutations in Fumarate Hydratase in Multiple Cutaneous and Uterine Leiomyomatosis and Renal Cell Cancer

Alam

Olpin

Rowan

et al. 2005

The Journal of Molecular Diagnostics

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“…Both homologs form a stable homotetramer containing four active sites, and every active site is composed of residues from three enzyme subunits. Each dumbbell-shaped subunit within the tetramer contains three domains: an N-terminal domain, a central domain, and a C-terminal domain (8)(9)(10). The N-and C-terminal domains are predominantly α-helical and linked by the central domain that consists of five tightly packed helices.…”

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confidence: 99%

Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

Kasbekar

Fischer

Mott

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Enzymes in essential metabolic pathways are attractive targets for the treatment of bacterial diseases, but in many cases, the presence of homologous human enzymes makes them impractical candidates for drug development. Fumarate hydratase, an essential enzyme in the tricarboxylic acid (TCA) cycle, has been identified as one such potential therapeutic target in tuberculosis. We report the discovery of the first small molecule inhibitor, to our knowledge, of the Mycobacterium tuberculosis fumarate hydratase. A crystal structure at 2.0-Å resolution of the compound in complex with the protein establishes the existence of a previously unidentified allosteric regulatory site. This allosteric site allows for selective inhibition with respect to the homologous human enzyme. We observe a unique binding mode in which two inhibitor molecules interact within the allosteric site, driving significant conformational changes that preclude simultaneous substrate and inhibitor binding. Our results demonstrate the selective inhibition of a highly conserved metabolic enzyme that contains identical active site residues in both the host and the pathogen. Because the tricarboxylic acid (TCA) cycle connects many pathways of cellular metabolism, preventing the function of this cycle through enzyme inhibition is an attractive strategy for targeting infectious agents (1). In Mycobacterium tuberculosis, experimental evidence (2) has suggested that fumarate hydratase, the essential enzyme responsible for the reversible conversion of fumarate to (L)-malate, is a vulnerable target. This vulnerability is in part due to the fact that, unlike other bacteria (such as Escherichia coli), M. tuberculosis expresses only one enzyme that performs this function (3). In addition to its role in metabolism under aerobic conditions, fumarate hydratase has also garnered interest because of the discovery of a flux toward the reverse TCA cycle under hypoxic conditions in nonreplicating M. tuberculosis (2, 4, 5). However, despite these discoveries, no small molecule inhibitor of the M. tuberculosis fumarate hydratase has been reported. The discovery of such an inhibitor would provide an important tool to begin probing the role of the TCA cycle in both actively replicating and nonreplicating bacteria.From the standpoint of drug development, however, targeting the M. tuberculosis fumarate hydratase poses a significant challenge, because the protein is highly evolutionarily conserved. In particular, the human and M. tuberculosis homologs share identical active site residues as well as 53% overall sequence identity (6, 7). Both homologs form a stable homotetramer containing four active sites, and every active site is composed of residues from three enzyme subunits. Each dumbbell-shaped subunit within the tetramer contains three domains: an N-terminal domain, a central domain, and a C-terminal domain (8-10). The N-and C-terminal domains are predominantly α-helical and linked by the central domain that consists of five tightly packed helices. The central domains of ...

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“…The recent three-dimensional structure of the fumarase C of Escherichia coli has identified a binding site for anions which is generated by side chains from three of the four subunits within the tetramer [2]. These same side chains are found in the three most highly conserved regions [3].…”

Section: Introductionmentioning

confidence: 99%

MD Simulation Studies of Fumarase Reveal Thermo Dynamical Stability

Singh¹

2016

J Phys Chem Biophys

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The multisubunit active site of fumarase C from Escherichia coli

Cited by 123 publications

References 36 publications

Missense Mutations in Fumarate Hydratase in Multiple Cutaneous and Uterine Leiomyomatosis and Renal Cell Cancer

Missense Mutations in Fumarate Hydratase in Multiple Cutaneous and Uterine Leiomyomatosis and Renal Cell Cancer

Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

MD Simulation Studies of Fumarase Reveal Thermo Dynamical Stability

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