Trapping of an Intermediate in the Reaction Catalyzed by Flavin-Dependent Thymidylate Synthase

Mishanina, Tatiana V.; Koehn, Eric M.; Conrad, John A.; Palfey, Bruce A.; Lesley, Scott A.; Kohen, Amnon

doi:10.1021/ja2120822

Cited by 33 publications

(69 citation statements)

References 24 publications

(76 reference statements)

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“…Furthermore, in contrast to the protein methylation suggested in ref. 16, we were unable to identify any methylene transfer to the protein using radiolabeled (8). These findings, along with multiple kinetic data (10, 13-15, 18-20, 27), encouraged the examination of an alternative CH 2 H 4 folate binding mode that would enable direct methylene transfer to dUMP.…”

Section: Discussionmentioning

confidence: 91%

“…2). (4,8). (B) A mechanism proposed for methylene transfer that involves an enzymatic arginine residue (16).…”

Section: Resultsmentioning

confidence: 99%

“…This observation led to the identification of alternative flavin-dependent thymidylate synthases (FDTSs), which are encoded by the thyX gene and have no sequence or structure homology to classic TSase enzymes (2,6,7). Furthermore, multiple studies have identified key differences in the molecular mechanism of catalysis between FDTSs and classic TSases (2,4,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). These differences, along with the fact that the thyX gene is present in many human pathogens (e.g., bacteria causing Anthrax, Tuberculosis, Typhus, and other diseases), renders these flavo-enzymes as potential targets for rational inhibitor design, possibly affording compounds that might be effective antimicrobial drugs (11,(22)(23)(24).…”

mentioning

confidence: 99%

“…The FAD can be reduced to FADH 2 by nicotinamides, ferrodoxin, and other small molecule reductants (e.g., dithionite). Although the reductive halfreaction is activated by dUMP the conversion of dUMP to dTMP occurs during the oxidative half-reaction (FADH 2 →FAD) (8,10,14,18,27). In contrast to classic TSase, where the cofactor CH 2 H 4 folate provides both the H − and methylene, in the FDTS reaction the H − is provided by the FADH 2 and CH 2 H 4 folate is used only as a source for the methylene moiety (18,20,28).…”

mentioning

confidence: 99%

“…1A) from CH 2 H 4 folate to dUMP (4,8,10,(13)(14)(15)(18)(19)(20), or transfer of the methylene via an arginine residue (Arg174 in Thermatoga maritima FDTS; TmFDTS) before its delivery to dUMP (Fig. 1B) (16).…”

mentioning

confidence: 99%

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Folate binding site of flavin-dependent thymidylate synthase

Koehn

Perissinotti

Moghram

et al. 2012

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

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The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH 2 H 4 folate). Most organisms, including humans, rely on the thyA-or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs.

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Section: Discussionmentioning

confidence: 91%

“…2). (4,8). (B) A mechanism proposed for methylene transfer that involves an enzymatic arginine residue (16).…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Folate binding site of flavin-dependent thymidylate synthase

Koehn

Perissinotti

Moghram

et al. 2012

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

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Nucleosides Modified at the Base Moiety

Sari

Roy

Agrofoglio

2013

Chemical Synthesis of Nucleoside Analogues

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Desolvation Induced Conformational Transition in a Cryptophane Host: Crystal To Crystal Transformation

Ghosh,

Hossain,

Saradhi Satha

et al. 2023

Chemistry An Asian Journal

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Cryptophanes show different conformations in solution and solid state depending upon various factors, such as the length of connecting linkers, medium, and nature of the incoming guest molecule(s). A cyclotriguaiacylenes (CTG) based cryptophane molecule was synthesized using click chemistry containing three triazole linkers and studied as well. This molecule shows two conformations, out-out crown-crown (CC), and out-in CC, in the presence or absence of guest molecule(s), as studied both in solution and solid state. The out-in CC, in which both CTG fragments are in crown conformation with one crown sitting above the other, could be obtained by slow escape of the trapped acetone molecules from out-out CC in solid state. This transformation could be obtained through a single-crystal-to-single-crystal (SCSC) transformation from a large volume out-out CC to a smaller volume out-in CC conformation which is also supported by density functional theory calculations.Cavitands are organic molecules with enforced cavity large enough to encapsulate various small molecule(s). [1] The interior cavity of cavitands have been used for stabilization of unstable molecules, [2,3] trapping of reaction intermediate(s), [1e,4] visible light photocatalysis, [5] asymmetric catalysis, [6] enantioselective recognition, [7] for stabilizing reactive intermediates, [7] as microreaction chambers, [8,9] chlorine capture with attomolar affinity, [10] modulate chiroptical properties. [11] Cryptophanes, are one such class of molecules, [12] contain two bowl shaped cyclic molecular systems (cyclotribenzylenes or its derivatives), joined together with three linkers. Changing linker size changes the free space of the cavity considerably. [13,14] Cryptophanes are well studied for entrapment of small neutral guest molecules CHCl 3 , CH 2 Cl 2 , CHFClBr, [13a] cationic organic molecules, [15,16] metal ions, [17] and gases like Xe, CFCs, CH 4 in solution phase. [13,18] These molecules are shown to bind biologically important molecules such as choline and avidin. [16] Typically, cryptophanes remain in an outout CC conformation, in which the connecting methylene groups in the cryptophane unit pointed oppositely outward. However, when flexible enough, i. e., the connecting alkyl chain contains 6 or more carbons, one of the cyclotribenzylenes (CTBs) unit flips to give an out-in CC conformation (Scheme 1).In cryptophanes, one of the factors that governs the feasibility of host-guest complexation is length of the linker. Thus, with an optimise linker length, small molecule can trigger the conversion of out-out CC to out-in CC or vice-versa. Such stimuli-responsive materials have potential applications in sensors, actuators, photoelectronic devices, information storage etc. [19] When changes occur due to escape of the trapped guest molecule(s), compresses the crystals and such systems are proposed to be helpful in designing the high-energy molecular actuators. [20] Out-out CC conformer in cryptophanes is generally found in solid state with small...

show abstract

Trapping of an Intermediate in the Reaction Catalyzed by Flavin-Dependent Thymidylate Synthase

Cited by 33 publications

References 24 publications

Folate binding site of flavin-dependent thymidylate synthase

Folate binding site of flavin-dependent thymidylate synthase

Nucleosides Modified at the Base Moiety

Desolvation Induced Conformational Transition in a Cryptophane Host: Crystal To Crystal Transformation

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