Literature Report: Folate Induced Conformational Change for Methyl Transfer Speaker: Chen-Yang ZHOU Supervisor: Yun-Dong WU Google’s home page in 5.12: Dorothy Mary Hodgkin Dorothy Mary Hodgkin, born in May 12, 1910 The 3rd woman to win the Nobel Prize in Chemistry (1964), for confirmation of the structure of penicillin and the Vitamin B12 using X-ray crystallography What is metalloprotein Metal + Protein = Metalloprotein Protein containing a metal ion cofactor. Estimated that ~ 1/2 of all proteins contain a metal. Outline • Example of 2 important reactions that matalloproteins play a central role • The advantage of metals • 1 detailed example: the importance of protein Example 1: Photosynthesis 6CO2 + 12 H2O light Oxygen evolution: 2H2O C6H12O6 + 6O2 +6H2O light 4e- + 4H+ + O2 • Photosynthesis is the most important reaction in our planet • CO2 is converted into sugar, and generate O2 • Photosynthesis fixes ~1011 tons of carbon per year • Oxygen evolution step is first key step, with metallo-oxo cluster as catalytic center • Metallo-oxo cluster comprising 4 Mnn+ (n from 3 to 5) and 1 Ca • Structure is controversial • Cubane like, 2011, 1.9Å Yasufumi Umena, Keisuke Kawakami, Jian-Ren Shen & Nobuo Kamiya. Nature 2011; 473: 55-60 Example 2: N2 Fixation • • • • Nitrogen fixation: N2 the atmosphere is converted into ammonia NH3 In industry: Haber-Bosch Process, high temperature and pressure In Nature: Nitrogenase 1.16Å structure resolved in 2002, but identification to the atom in the center is still uncertain. (until 2009) Rhizobium（根瘤菌） MoFeS-containing cofactor: breaking the N,N triple bond Einsle O, Tezcan FA, Andrade SLA, Schmid B, Yoshida M, et al. 2002. Science 297:1696–700 What’s the advantage of metals • Metallocofactors can bind gases • Transition metals accept lone pair that act Lewis Acids • Form coordination complex Red blood cell Myoglobin What’s the advantage of metals • Metallocofactors can act as supernucleophiles SN2 reaction mechanism Cob(I)alamin(vitamin B12): supernucleophile What’s the advantage of metals • Radical-based reaction: biotin synthase Dethiol biotin Biotin • Radical-based reaction: halogenation by SyrB2 Blasiak, L. C., Vaillancourt, F. H., Walsh, C. T. and Drennan, C. L. Nature 2006, 440, 368–371 We’ve seen metal play a central role, why protein is necessary? Detailed Example: Acetogenesis • Carbon Fixation Without Oxygen • Acetogenesis fixes ~1010 tons of carbon per year, 10% of total • One of the six known pathways for carbon fixation (Wood-Ljungdahl pathway) • Energy efficient: lowst net ATP requirement • O2-sensitive: Fe-S, Ni-Fe-S clusters (anaerobic) Acetogenesis: Wood-Ljungdahl pathway CH3H4folate CO2 CO Metallo-centers in Acetogenesis • Key to acetogenesis are protein-bound NiFeS-containing and Co-containing cofactors Binds and transfers CH3 Binds and reduces CO2 to CO Assembles the acetate S.W. Ragsdale, E. Pierce, Biochimica et Biophysica Acta 2008; 1784: 1873–1898 (Review) Zoom in to the Methyl Transfer Step Vitamine B12 derivative, Cobalamin, is a supernucleophile They form a complex Methyl-transferase Corrinoid FeS Protein Protein Structures in Methyl Transfer Step 83kDa Why such an elaborate protein framework (140kDa) is required for such a simple, yet biologically essential reaction? 56kDa Ando N, Kung Y, Can M, Bender G, Ragsdale SW, Drennan CL. (2012) J. Am. Chem. Soc. 134 (43) 17945–17954. Protein Structures in Methyl Transfer Step The distance for the reactant is ~25Å Swinging move ~7Å towards the CH3 1. B12 must be uncapped to do SN2 substitution, and move a distance of ~18Å 2. Is the crystal structure active? Is the Crystal active? Different valance state of Co has different color control group Kung Y, et al. 2012. Nature 484:265–269. Result of ultraviolet– visible absorption spectroscopy Folate induce clamping motion: ~17Å Resolution of crystal structures Folate-free: 2.38 Å Folate-bound: 3.50 Å Kung Y, et al. 2012. Nature 484:265–269. The last move of B12 Intriguingly, a large, continuous electron density peak is present in 2Fo − Fc, Fo − Fc, and composite omit maps, emanating from the corrin ring and stretching directly over the folate-binding site, 2Fo-Fc map in blue mesh (1.0 σ) Fo-Fc difference map in green mesh (3.0 σ) Anomalous difference map in pink mesh (4.0 σ) Cartoon model of B12-dependent methyl transfer in CFeSP/MeTr Kung Y, et al. 2012. Nature 484:265–269. Summary • Protein protect the reactive B12 by capping domain before and after CH3 transfer • The conformational change is controlled by protein, which send the B12 to the CH3-H4folate. • The crystal complex is active, and the conformational change can happen even in the crystal lattice. (The largest conformational change observed in crystal.) • Protein scaffold and metallo center depend on each other to have amazing reactivity and selectivity Thank you!