On Transition Structures for Hydride Transfer Step: A Theoretical Study of the Reaction Catalyzed by Dihydrofolate Reductase Enzyme

Andrés, Juán; Moliner, Vicent; Safont, Vicent S.; Domingo, Luís R.; Picher, M. Teresa; Krechl, Jiří

doi:10.1006/bioo.1996.0002

Cited by 32 publications

(49 citation statements)

References 37 publications

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“…2) Additional important interactions include the hydrogen bonded Y69-Q67-Q67′-Y69′ surfaces which sandwich the two ring systems in an endo orientation, several additional hydrogen bonds, particularly with the nicotinamide ribose, which help to tilt the ring systems, electrostatic interactions with the K32 residues in the pore, and finally stacking of the nicotinamide and pteridine rings with each other, which also helps to explain the cooperativity of binding (9). The tilted, endo ring orientation closely approaches the theoretical transition state modeled by Andres et al (31). The reliance on critical backbone interactions for optimal positioning provides a mechanism that relieves the evolutionary problem caused by the inability of the enzyme to mutate individual active site residues in order to optimize binding and activity.…”

Section: Discussionsupporting

confidence: 69%

“…The ribonicotinamide is also positioned by a network of H-bonds that involves residues from chains A and B (Figure 5a). These interactions not only hold the nicotinamide ring in position for hydride transfer for the DHF, but also tilt the ring so that the reactive centers on the DHF and nicotinamide ring adopt a geometry that more closely approximates that of an endo transition state as modeled by Andres et al (31). Additionally, two Lys32 residues (chains A and D) interact with the NADP + cofactor (Figure 5b).…”

Section: Substrate/cofactor Recognitionmentioning

confidence: 86%

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

“…This ring stacking geometry is not observed in the Type I dihydrofolate reductase ( Figure 6b), but has recently been observed in pteridine reductase (40) (Figure 6c), although in that case, the nicotinamide ring is flipped, leading to a B-side hydride transfer. Theoretical studies have suggested that this relative orientation may be optimally efficient for hydride transfer (31,(41)(42)(43).In type I DHFR, a carboxylate sidechain from a glutamate (mammalian) or an aspartate (bacterial) sidechain is positioned to hydrogen bond with the pteridine N-3 and with the exo amino group of the pteridine ring (Figure 6b) (27,44). In pteridine reductase, the adenosyl-5′-phosphate group is analogously positioned near the pteridine N3 (Figure 6c).…”

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confidence: 99%

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Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

et al. 2007

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Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the Dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR•DHF•NADP + catalytic complex, resolved to1.26 Å. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67′-Y69′ residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationallychallenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation. KeywordsR67 DHFR; Dihydrofolate Reductase; X-ray crystallography; ternary complex; Type II DHFR Antifolate drug therapy plays a critical role in the treatment of pathogenic and neoplastic diseases. The evolution of a plasmid-encoded, Type II dihydrofolate reductase (DHFR) provides one mechanism for bacterial evasion of drugs such as trimethoprim that target the bacterial dihydrofolate reductase enzyme (1-4). Type II DHFR is an extremely unusual enzyme that exhibits no apparent structural or evolutionary relationship with the type I (chromosomal) enzyme. It is one of the smallest enzymes known to self-assemble into an active quaternary structure, forming a homotetramer consisting of four 78-residue peptides * To whom correspondence should be addressed. This type of active site structure also creates substantial evolutionary and mutational challenges to the enzyme. Since each mutation will alter four active site residues at a time, most of the evolutionary pressure that would normally optimize enzyme function is compromised by the need to balance the effects of substitutions at all four symmetry-related sites. For example, a residue substitution on one monomer, which might promote folate N5 protonation, may also interfere with NADPH binding when it is prese...

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Section: Discussionsupporting

confidence: 69%

Section: Substrate/cofactor Recognitionmentioning

confidence: 86%

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

et al. 2007

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“…At least two different transition states are available to the DHFR reaction, [87] as illustrated in Figure 5. Constraints imposed by the Ec chromosomal DHFR structure lead to its using an exo transition state with minimal overlap of the pteridine and nicotinamide rings.…”

Section: Transition States?mentioning

confidence: 99%

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Howell

2005

ChemBioChem

117

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There are numerous examples of proteins that catalyze the same reaction while possessing different structures. This review focuses on two dihydrofolate reductases (DHFRs) that have disparate structures and discusses how the catalytic strategies of these two DHFRs are driven by their respective scaffolds. The two proteins are E. coli chromosomal DHFR (Ec DHFR) and a type II R-plasmid-encoded DHFR, typified by R67 DHFR. The former has been described as a very well evolved enzyme with an efficiency of 0.15, while the latter has been suggested to be a model for a "primitive" enzyme that has not yet been optimized by evolution. This comparison underlines what is important to catalysis in these two enzymes and concurrently highlights fundamental issues in enzyme catalysis.

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Evolution of a Transition State: Role of Lys100 in the Active Site of Isocitrate Dehydrogenase

et al. 2014

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An active site lysine essential to catalysis in isocitrate dehydrogenase (IDH) is absent from related enzymes. As all family members catalyze the same oxidative β-decarboxylation at the 2R-malate core common to their substrates, it seems odd that an amino acid essential to one is not found in all. Ordinarily, hydride transfer to a nicotinamide C4 neutralizes the positive charge at N1 directly. In IDH the negatively charged C4-carboxylate of isocitrate stabilizes the ground state positive charge on the adjacent nicotinamide N1, opposing hydride transfer. The critical lysine is poised to stabilize – and perhaps even protonate – an oxyanion formed on the nicotinamide 3-carboxamide, thereby enabling the hydride to be transferred while the positive charge at N1 is maintained. IDH may catalyze the same overall reaction as other family members, but dehydrogenation proceeds through a distinct, though related, transition state. Partial activation of lysine mutants by K+ and NH4+ represents a throwback to the primordial state of the first substrate promiscuous family member.

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On Transition Structures for Hydride Transfer Step: A Theoretical Study of the Reaction Catalyzed by Dihydrofolate Reductase Enzyme

Cited by 32 publications

References 37 publications

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Evolution of a Transition State: Role of Lys100 in the Active Site of Isocitrate Dehydrogenase

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