1. Ultrafast Dynamics of Fully Reduced Flavin in Catalytic Structures of Thymidylate Synthase ThyX
- Author
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Murielle Lombard, Fabien Lacombat, Pascal Plaza, Djemel Hamdane, Nadia Dozova, Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie des Processus Biologiques (LCPB), and Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Dinitrocresols ,General Physics and Astronomy ,Flavin group ,Molecular Dynamics Simulation ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Thymidylate synthase ,Cofactor ,03 medical and health sciences ,chemistry.chemical_compound ,Thermotoga maritima ,Physical and Theoretical Chemistry ,Methylene ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,030304 developmental biology ,Flavin adenine dinucleotide ,0303 health sciences ,biology ,Molecular Structure ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Substrate (chemistry) ,Thymidylate Synthase ,Conical intersection ,0104 chemical sciences ,[CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry ,chemistry ,biology.protein ,Biocatalysis ,Oxidation-Reduction ,Methyl group - Abstract
International audience; Thymidylate is a vital DNA precursor synthesized by thymidylate synthases. ThyX is a flavin-dependent thymidylate synthase found in several human pathogens and absent in humans, which makes it a potential target for antimicrobial drugs. This enzyme methylates the 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate (dTMP) using a reduced flavin adenine dinucleotide (FADH-) as prosthetic group and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF) as a methylene donor. Recently, it was shown that ThyX-catalyzed reaction is a complex process wherein FADH- promotes both methylene transfer and reduction of the transferred methylene into a methyl group. Here, we studied the dynamic and photophysics of FADH- bound to ThyX, in several substrate-binding states (no substrate, in the presence of dUMP or folate or both) by femtosecond transient absorption spectroscopy. This methodology provides valuable information about the ground-state configuration of the isoalloxazine moiety of FADH- and the rigidity of its local environment, through spectra shape and excited-state lifetime parameters. In the absence of substrate, the environment of FADH- in ThyX is only mildly more constrained than that of free FADH- in solution. The addition of dUMP however narrows the distribution of ground-state configurations and increases the constraints on the butterfly bending motion in the excited state. Folate binding results in the selection of new ground-state configurations, presumably located at a greater distance from the conical intersection where excited-state decay occurs. When both substrates are present, the ground-state configuration appears on the contrary rather limited to a geometry close to the conical intersection, which explains the relatively fast excited-state decay (100 ps on the average), even if the environment of the isoalloxazine is densely packed. Hence, although the environment of the flavin is dramatically constrained, FADH- retains a dynamic necessary to shuttle carbon from folate to dUMP. Our study demonstrates the high sensitivity of FADH- photophysics to the constraints exerted by its immediate surroundings.
- Published
- 2021
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