Crystallographic Studies of the Catalytic and a Second Site in Fumarase C from <i>Escherichia coli</i><sup>,</sup>

Weaver, Todd; Banaszak, Leonard J.

doi:10.1021/bi9614702

Cited by 93 publications

(151 citation statements)

References 35 publications

(58 reference statements)

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“…4,8,23,26 Similar roles have been proposed for the equivalent histidine/ glutamate residues in ASL/δ2-crystallin 8,27-29 and fumarase C. 4,30 Mutation of H171 with a variety of substitutions (e.g. A, L, E, Q, R) in bs-ADL has highlighted the importance of this residue in catalysis, as residual activity was only detected in the H171R mutant, which was found to be 10 5 -fold less active than the wild-type enzyme.…”

Section: Resultsmentioning

confidence: 61%

“…This residue is strictly conserved across the superfamily and has been suggested to play a role in stabilizing the aci-carboxylate intermediate in several of the superfamily members. 12,13,30 This proposal is supported by mutagenesis experiments in E. coli L-aspartase 13 and dδc2 (M. T., unpublished work), and by the dδc1-sulfate complex, which demonstrated that closure of the C3 loop over the active site enabled the N ζ atom of the equivalent lysine residue to interact with the sulfate ion. 33 However, in the H171A-ADS complex K301 is not in a position to interact with the δ-carboxylate group of the substrate, on which the negative charges accumulate in the aci-carboxylate intermediate.…”

Section: Stabilization Of the Aci-carboxylate Intermediatementioning

confidence: 92%

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Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

Tsai

Koo

et al. 2007

Journal of Molecular Biology

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Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimida-zole-(Nsuccinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/ fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly α-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall β-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP•FUM) complexes have been determined to 2.0, 1.85, and 2.0 Å resolution, respectively. The H171A-ADS and H171N-AMP•FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287-303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observed in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.

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Section: Resultsmentioning

confidence: 61%

Section: Stabilization Of the Aci-carboxylate Intermediatementioning

confidence: 92%

Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

Tsai

Koo

et al. 2007

Journal of Molecular Biology

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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.…”

mentioning

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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“…E. coli fumarase is unrelated to CM in sequence or function but, like CM, is largely ␣-helical (24). To evaluate in a broad and unbiased manner the ability of foreign protein fragments to substitute regions of CM, fumarase was recombined with mMjCM by using protein NRR.…”

Section: Recombination With An Unrelated Protein Results In Active Chmentioning

confidence: 99%

Directed evolution of protein enzymes using nonhomologous random recombination

Bittker

Liu

et al. 2004

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

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We recently reported the development of nonhomologous random recombination (NRR) as a method for nucleic acid diversification and applied NRR to the evolution of DNA aptamers. Here, we describe a modified method, protein NRR, that enables proteins to access diversity previously difficult or impossible to generate. We investigated the structural plasticity of protein folds and the ability of helical motifs to function in different contexts by applying protein NRR and in vivo selection to the evolution of chorismate mutase (CM) enzymes. Functional CM mutants evolved using protein NRR contained many insertions, deletions, and rearrangements. The distribution of these changes was not random but clustered in certain regions of the protein. Topologically rearranged but functional enzymes also emerged from these studies, indicating that multiple connectivities can accommodate a functional CM active site and demonstrating the ability to generate new domain connectivities through protein NRR. Protein NRR was also used to randomly recombine CM and fumarase, an unrelated but also ␣-helical protein. Whereas the resulting library contained fumarase fragments in many contexts before functional selection, library members surviving selection for CM activity invariably contained a CM core with fumarase sequences found only at the termini or in one loop. These results imply that internal helical fragments cannot be swapped between these proteins without the loss of nearly all CM activity. Our findings suggest that protein NRR will be useful in probing the functional requirements of enzymes and in the creation of new protein topologies. D irected evolution has been used to alter (1-4) enzyme activity as well as to investigate sequence constraints on protein folding (5-7) and catalysis (8). The sequences and therefore the properties of proteins that emerge from directed evolution depend on the diversification method used to generate the protein library before screening or selection. Theoretical studies (9) suggest that diversification methods, including point mutagenesis, homologous recombination, secondary structure swapping, and nonhomologous recombination differ in their ability to access protein sequences representing new folds and improved functions. Of the methods listed above, nonhomologous recombination has been theorized to be the most effective at enabling new structures and functions to emerge during protein evolution. Additional studies on enzyme superfamilies suggest that the reorganization of structurally similar components can result in altered substrate specificity or function (10). Because these components often lack DNA sequence homology, their combinatorial diversification can in general only be accomplished through nonhomologous recombination.Methods that enable recombination to take place at defined sites without sequence homology have been recently described (11). For example, it is possible to recombine unrelated proteinencoding genes by using synthetic oligonucleotides to encode each desired crossover (12, 1...

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Crystallographic Studies of the Catalytic and a Second Site in Fumarase C from Escherichia coli^,

Cited by 93 publications

References 35 publications

Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

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

Directed evolution of protein enzymes using nonhomologous random recombination

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Crystallographic Studies of the Catalytic and a Second Site in Fumarase C from Escherichia coli,

Cited by 93 publications

References 35 publications

Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

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

Directed evolution of protein enzymes using nonhomologous random recombination

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Product

Resources

About

Crystallographic Studies of the Catalytic and a Second Site in Fumarase C from Escherichia coli^,