HAL Id: hal-02075703
https://hal.univ-lorraine.fr/hal-02075703
Submitted on 21 Mar 2019
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Enzyme Active Site Loop Revealed as Gatekeeper for Cofactor Flip by Targeted Molecular Dynamics
Simulations and FRET-kinetics
Sophie Rahuel-Clermont, R Bchini, Sophie Barbe, S Boutserin, I. Andre, F Talfournier
To cite this version:
Sophie Rahuel-Clermont, R Bchini, Sophie Barbe, S Boutserin, I. Andre, et al.. Enzyme Active Site Loop Revealed as Gatekeeper for Cofactor Flip by Targeted Molecular Dynamics Simulations and FRET-kinetics. Molecular Biophysics: ABC of the puzzle of Life ARBRE-MOBIEU Plenary Meeting., Mar 2019, Zagreb, Croatia. �hal-02075703�
Trp177 8.3 Å
19.5 Å
Sophie Rahuel-Clermont
1, Raphaël Bchini
1, Sophie Barbe
2, Isabelle André
2, François Talfournier
11
Laboratoire IMoPA, Equipe Enzymologie Moléculaire et Structurale, UMR 7365 CNRS-Université de Lorraine, Vandœuvre-lès-Nancy France;
2Molecular Modelling & Design, Catalysis & Enzyme Molecular Engineering,
Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), UMR INSA - CNRS 5504 - INRA 792, Institut National des Sciences Appliquées, Toulouse, FRANCE
Enzyme Active Site Loop Revealed as Gatekeeper for Cofactor Flip by Targeted Molecular Dynamics Simulations and FRET-kinetics
GAPN RALDH2
D’Ambrosio et al., 2006, Biochemistry, 45 : 2978-86
NAD(P)H
Excitation 295 nm
HN CH C CH2
O
HN
Emission 330 nm
Emission 450 nm N
R
O
NH2 H H
The context
Aldehyde dehydrogenases: NAD(P)-dependent dehydrogenases
Involved in detoxication and metabolic pathways (retinoic acid biosynthesis)
NADPH nicotinamide moiety
NADP+ in hydride
transfer conformation
NADPH in hydrolysis conformation
Reduced nicotinamide moiety (NMNH) flipping is required for Glu-268 to activate the water molecule in the deacylation step
Glu-268
Glu-268 activates H2O in hydrolysis
The question
Driving force?
Pathway?
The strategy
Reduced nicotinamide moiety (NMNH) flipping
Impact on catalysis ?
→ Kinetic approach
→ Intrinsic Trp/NADH FRET probe
Driving force ?
→ Identification of molecular factors allowing cofactor movement
→ Targeted Molecular Dynamics
Wavelength, nm
Fluorescence intensity
NMNH conformation monitoring by FRET
Enzyme Trp emission Enzyme-NADH complex emission
NADH emission
excitation @ 295 nm (Trp residues)
Rapid kinetic monitoring of the flip using stopped flow
Acylation = NADH formation in hydride transfer conformation
Deacylation=
NADH released from active site
3
2
Viscogen effect(glycerol/saccharose)
→ conformational transition = FLIP
1
Conserved ELGG loop as a gatekeper for cofactor flipping
Targeted Molecular Dynamics: GAPN
hydride transfer hydrolysis
Trp177 is the specific probe of cofactor flipping
G271A mutation in ELGG loop drastically reduces the flip kinetics
Thioacylenzyme intermediate
NADH NAD+
Pyruvate Lactate
LDH (kobs ~ 130 s-1)
+ LDH/pyruvate coupled assay
→ simplifies kinetics by bleaching the free NADH
3
RALDH2 G271A
- acylation remains non rate-limiting - → allows flip to be measured
- ↘↘ of the flip rate constant
k
flip(s
-1) k
cat(s
-1)
WT 5 2
G271A 0,03 0,01
GAPN
RALDH2
RALDH2 WT
▪ Flip/catalytic efficiency relies on ELGG loop flexibility
▪ Cofactor flipping extends to all ALDH family including CoA- dependent aldehyde dehydrogenases
Hypothesis
Role of E
268LGG
271loop in flip control ?
➔ Mutation Gly271Ala
2
Rate-limiting FLIP
MSDH
No significant change of the protein structure
E268 L269
G270
G271
K306
E399
F401
thioacyl enzyme
NADPH C302
E
268LGG
271loop
For MSDH only*
thioacylenzyme
Representative trajectory
ELGG loop
+ LDH/pyruvate
+ glycerol 20%
- glycerol
2 FLIP
hydrolysis conformation
hydride transfer conformation
2
CONCLUSION
References
D’Ambrosio, K., Pailot, A., Talfournier, F., Didierjean, C., Benedetti, E., Aubry, A., Branlant, G., and Corbier, C. (2006) The First Crystal Structure of a Thioacylenzyme Intermediate in the ALDH Family: New Coenzyme Conformation and Relevance to Catalysis. Biochemistry. 45, 2978–2986
Rahuel-Clermont, S., Bchini, R., Barbe, S., Boutserin, S., André, I., and Talfournier, F. (2019) Enzyme Active Site Loop Revealed as a Gatekeeper for Cofactor Flip by Targeted Molecular Dynamics Simulations and FRET-Based Kinetics. ACS Catal. 9, 1337−1346
Cofactor RMSD (Å)
RalDH2 pdb 3SBC
