GLYCOSIDIC BONDS? an Ab Initio Molecular Dynamics Investigation

HOW DOES NATURE FORM GLYCOSIDIC BONDS? An ab initio molecular dynamics investigation

Carme RiRovira Universitat de Barcelona – Parc Científic de Barcelona Carbohydrates

50% of our daily calorie intake comes from carbohydrates

http://en.wikipedia . org/

Carbohydrates are our “biological fuel “, as well as the primary form of storage and energy consumption in organisms Introduction The roles of carbohydrates

PlPolysacch arid es starch cellulose . Structural support . EtEnergy storage

Glycoconjugates . Cell-cell interaction . Signal transduction . Immune response

. Parasitic infections carbohydrates GlcNAcMan GlcNAc http://www.glycomicscentre.ca 5 2

DihiDeciphering mechihanisms in whic h carbhdbohydrates are ilidimplicated is of enormous interest for the search of new therapeutic agents. Introduction Glicosidic bond

OH glucose OH glucose glucose glucose O O HO HO OH OH O OH O O O O OH O HO OH O

HO HO glycosidic bond: C-O bond between two sugar units How do glycosidic bonds form?

Most glycosidic bonds are synthesized in nature from sugars that are activated by a cofactor

Enzyme (glycoside transferase) How do glycosidic bonds form?

The ggylycosidic bond is formed u pon transfer of a su gar molecule from the donor (an activated sugar) to an acceptor molecule (typically another sugar)

Enzyme (glycoside transferase) Two modes of enzyme operation

Retention or inversion of the configuration of the anomeric carbon

retiitaining GT

The molecular mechanism of retaining GTs is very controversial

Palcic, Curr. Opin. Chem. Biol. 2011; Lee et al. Nat. Chem. Biol. 2011 Lairson et al. Annu. Rev. Biochem. 2008 Retention of the configuration of the anomeric carbon

retiitaining GT

high steric hindrance is expected Possible mechanism for retaining enzymes

covalent glycosyl-enzyme intermediate Possible mechanism for retaining enzymes

covalent glycosyl-enzyme intermediate (double displacement, ~ retaining GHs)

retaining GHs e.g. Biarnés et al. J. Am. Chem. Soc. 133, 20301–09, 2011 Possible mechanism for retaining enzymes

covalent glycosyl-enzyme intermediate (~ retaining GHs)

But

• All experimental attempts to isolate a glycosyl-enzyme intermediate have failed • Few GTs have a putative Another possibility

+ The reaction takes place on - a single “face” of the sugar “front-face attack”

BtBut • High steric hindrance expected • Little chemical precedence Controversy

Two covalent bonds being + broken/formed in the - same region of the space

• Is the ftfront-face reaction fiblfeasible? Simulation model

•Ab initio molecular dynamics (to take into account the atomic QM and electronic motion at room temperature) • QM/MM MM (Density Functional Theory/ AMBER)

• Metadynamics (Laio and Parrinello, PNAS 99, 12562-66, 2002) (to model the chemical reaction) Enzyme studied: trehalose-6-phosphate synthase

OH HO O OH OH OH OPO 2- HO O O HO O 3 OH HO O P P HO enzyme OPO 2- - O O 3 O O- O O OH OH HO HO O N O OH OH O + O O + HO HN O P P OH OH - O O- O O O O N HN glucose-6P trehalose-6P UDP O UDP-glucose (donor) (acceptor)

Enzyme Trehalose is a natural disaccharide used as food ingredient for its sweet flavor and preservative properties Enzyme·substrate complex

• Struct ure stbltableunder molllecular didynamics • Good agreement with binary complexes structures (Enzyme + UDP -Glc and Enzyme + UDP + Glc-6P) Free energy landscape

~ 100 QM atoms metastable intermediate 20 ps AIMD, 105 h MN (64/128 procs).

lifetime ~ 2 ps

(dos milésimas de una milmillonésima de segundo!) cleavage of phosphate-sugar bond R

1 R 3 glycosidic bond 2 4 formation

proton transfer Molecular mechanism of the front-face reaction

metadynamics trajectory

Glucose-6P

UDP

Theory: Ardèvol & Rovira, Angew. Chem. Int. Ed. 50, 10897 –901, 2011 Experiment: Seung et al. Nat. Chem. Biol. 7, 631-38, 2011

Acknowledgments

Albert Ardèvol (ETH, Switzerland)

Discussions with: Antoni Planas (Universitat Ramon Llull, Barcelona) Seung Lee, Ben Davis (University of Oxford, UK)