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Substrate activation in flavin-dependent thymidylate synthase.

Mishanina TV, Corcoran JM, Kohen A - J. Am. Chem. Soc. (2014)

Bottom Line: The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors.Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques.Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Iowa , Iowa City, Iowa 52242-1727, United States.

ABSTRACT
Thymidylate is a critical DNA nucleotide that has to be synthesized in cells de novo by all organisms. Flavin-dependent thymidylate synthase (FDTS) catalyzes the final step in this de novo production of thymidylate in many human pathogens, but it is absent from humans. The FDTS reaction proceeds via a chemical route that is different from its human enzyme analogue, making FDTS a potential antimicrobial target. The chemical mechanism of FDTS is still not understood, and the two most recently proposed mechanisms involve reaction intermediates that are unusual in pyrimidine biosynthesis and biology in general. These mechanisms differ in the relative timing of the reaction of the flavin with the substrate. The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors. Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques. Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data. These findings and the newly proposed mechanism add an important piece to the puzzle of the mechanism of FDTS and suggest a new class of intermediates that, in the future, may serve as targets for mechanism-based design of FDTS-specific inhibitors.

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Single-turnoverFDTS reaction kinetics overlaid with stopped-flowflavin absorbance trace (green, this work). Reduced flavin (FADH2) has no 420 nm absorbance, while oxidized flavin (FAD) does.Adapted with permission from ref (11).
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fig1: Single-turnoverFDTS reaction kinetics overlaid with stopped-flowflavin absorbance trace (green, this work). Reduced flavin (FADH2) has no 420 nm absorbance, while oxidized flavin (FAD) does.Adapted with permission from ref (11).

Mentions: In an alternative mechanism (Scheme 1b),dUMP is activated for the reaction with CH2H4fol via electronic polarization of the uracil moiety in the enzyme’sactive site upon binding. This dUMP polarization was proposed as theinitial step on the basis of the disappearance of dUMP in a single-turnoverexperiment occurring before flavin oxidation (ref (11) and the green trace inFigure 1). To the best of our knowledge, nosuch addition of formaldehyde and/or Mannich amines (analogous toiminium CH2H4fol) to the C5 of uracil has beenobserved before without the aid of a nucleophile, either in enzymesor in solution; nevertheless, such chemistry does not violate anyobvious chemical rules. Following elimination of H4folfrom the dUMP-folate adduct (step 3 in Scheme 1b), a positively charged exocyclic methylene intermediate would beobtained. This intermediate could then be reduced by FADH2 at C6 to yield the same isomer proposed in Scheme 1a, accounting for the observed D6 in the product dTMP.10 In such a mechanism, flavin oxidation happens after the methylene transfer; as a consequence, the reactionintermediates are not reduced, in sharp contrast to the mechanismin Scheme 1a.


Substrate activation in flavin-dependent thymidylate synthase.

Mishanina TV, Corcoran JM, Kohen A - J. Am. Chem. Soc. (2014)

Single-turnoverFDTS reaction kinetics overlaid with stopped-flowflavin absorbance trace (green, this work). Reduced flavin (FADH2) has no 420 nm absorbance, while oxidized flavin (FAD) does.Adapted with permission from ref (11).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4121000&req=5

fig1: Single-turnoverFDTS reaction kinetics overlaid with stopped-flowflavin absorbance trace (green, this work). Reduced flavin (FADH2) has no 420 nm absorbance, while oxidized flavin (FAD) does.Adapted with permission from ref (11).
Mentions: In an alternative mechanism (Scheme 1b),dUMP is activated for the reaction with CH2H4fol via electronic polarization of the uracil moiety in the enzyme’sactive site upon binding. This dUMP polarization was proposed as theinitial step on the basis of the disappearance of dUMP in a single-turnoverexperiment occurring before flavin oxidation (ref (11) and the green trace inFigure 1). To the best of our knowledge, nosuch addition of formaldehyde and/or Mannich amines (analogous toiminium CH2H4fol) to the C5 of uracil has beenobserved before without the aid of a nucleophile, either in enzymesor in solution; nevertheless, such chemistry does not violate anyobvious chemical rules. Following elimination of H4folfrom the dUMP-folate adduct (step 3 in Scheme 1b), a positively charged exocyclic methylene intermediate would beobtained. This intermediate could then be reduced by FADH2 at C6 to yield the same isomer proposed in Scheme 1a, accounting for the observed D6 in the product dTMP.10 In such a mechanism, flavin oxidation happens after the methylene transfer; as a consequence, the reactionintermediates are not reduced, in sharp contrast to the mechanismin Scheme 1a.

Bottom Line: The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors.Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques.Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Iowa , Iowa City, Iowa 52242-1727, United States.

ABSTRACT
Thymidylate is a critical DNA nucleotide that has to be synthesized in cells de novo by all organisms. Flavin-dependent thymidylate synthase (FDTS) catalyzes the final step in this de novo production of thymidylate in many human pathogens, but it is absent from humans. The FDTS reaction proceeds via a chemical route that is different from its human enzyme analogue, making FDTS a potential antimicrobial target. The chemical mechanism of FDTS is still not understood, and the two most recently proposed mechanisms involve reaction intermediates that are unusual in pyrimidine biosynthesis and biology in general. These mechanisms differ in the relative timing of the reaction of the flavin with the substrate. The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors. Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques. Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data. These findings and the newly proposed mechanism add an important piece to the puzzle of the mechanism of FDTS and suggest a new class of intermediates that, in the future, may serve as targets for mechanism-based design of FDTS-specific inhibitors.

Show MeSH