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Supplementary Discussion Comparison of protein barrel fold categories TIM barrel proteins (ie. the aldo-keto reductase family represented by pdb entry code 2ACQ) consist of a repeated -strand-loop--helix-loop motif, most often containing eight repeats, with the parallel -strands forming the interior of an open barrel, and the -helices forming the outer belt of the complete proteinS4 (Fig. S2a). More generally, -barrels consist of large structures (at least 200 amino acid residues in length), predominantly composed of alternating -helices and -strands, with parallel -strands forming a “hub” surrounded by a “tire” of -helicesS5. The class encompasses proteins containing mainly anti- -sheets but with segregated -helical and -sheet regions. In this regard, we propose that the -class definition, including protein domains exclusively composed of parallel -strands connected by -helices, should be enlarged to include Orf2’s novel architecture, the PT-barrel (Fig. S2b). Another protein structural class displaying an elliptical -barrel surrounded by helices is the dimeric ferredoxin-like sandwich fold. The ActVA-Orf6 monooxygenase (pdb entry code 1LQ9) from Streptomyces coelicolor A3 belongs to this structural class. This monooxygenase is a small dimeric enzyme that oxidizes a relatively large three ringed aromatic substrate in two active sites each of which is located between the -sheet and -helices of each monomer (Fig. S2c). -barrel proteins such as human fatty acid-binding protein (FABP, pdb entry code 1HMT), consist of ten anti-parallel -strands arranged as an elliptical barrel capped at the bottom by two short -helicesS6,S7 (Fig. S2d). Interestingly, a final type of protein barrel, the barrel, can be found in the -subunit of the heterodimeric human protein farnesyltransferase (PFTase), which catalyzes the carboxylterminal prenylation of Ras and several other signaling proteinsS8 (Fig. S2e). This domain displays a very different overall fold but presents a strikingly similar aromatic rich substrate binding pocket and active site topology as described for Orf2S9. Putative Orf2-mediated electrophilic geranylation mechanism of aromatic substrates A carbocation is proposed to result from the ionization of the diphosphate moiety, triggered by Mg2+ coordination, hydrogen bonds with Lys 119, Arg 228, Asn 173 and Lys 284, and cosubstrate binding. The positively charged C1 atom of the geranyl carbocation is now free to rotate toward the target double bond located 7 Å away on the prenyl acceptor. A “tyrosine belt” including Tyr 121, Tyr 175 and Tyr 216, surrounding the 10 carbons of GPP, may assist in the stabilization and positioning of the carbocationic intermediates via cation- interactionsS10. Upon capture of the reactive carbocation by the C5 atom of 1,6-DHN, a resonance stabilized carbocation or -complex formsS11. Tyr 216, which interacts with the diphosphate moiety of the GPP molecule, also forms a hydrogen bond to a well-ordered network of water molecules linked to the diphosphate moiety and located just above the aromatic substrate binding site. One of these water molecules is ideally positioned to abstract an acidic proton from the geranylated C5 atom of the -complex allowing for the restoration of the neutral aromatic product now containing a covalently tethered geranyl chain (Fig. S3). References S4. Gerlt, J.A. & Raushel, F.M. Evolution of function in (beta/alpha)8-barrel enzymes. Curr. Opin. Chem. Biol. 7, 252-264 (2003). S5. Branden, C.I. & Tooze, J. Introduction to protein structure. second ed. 1999, New York: Garland. S6. Sacchettini, J.C., Gordon, J.I. & Banaszak, L.J. Crystal structure of rat intestinal fatty-acidbinding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate. J. Mol. Biol. 208, 327-339 (1989). S7. Xu, Z., Bernlohr, D.A. & Banaszak, L.J. The adipocyte lipid-binding protein at 1.6 Å resolution. Crystal structures of the apoprotein and with bound saturated and unsaturated fatty acids. J. Biol. Chem. 268, 7874-7884 (1993). S8. Park, H.W. et al. Crystal structure of protein farnesyltransferase at 2.25 angstrom resolution. Science 275, 1800-1804 (1997). S9. Long, S.B., Casey, P.J. & Beese, L.S. Reaction path of protein farnesyltransferase at atomic resolution. Nature 419, 645-650 (2002). S10. Wise, M.L. & Croteau, R. in Comprehensive Natural Products Chemistry: Isoprenoids. D.E. Cane, Editor. 1998, Elsevier Science: Oxford, UK. S11. Olah, G.A. & Mo, Y.K. Stable carbocations. CXXXVI. Intramolecular 1,2-hydrogen shifts in difluoro- and dimethylbenzenium ions. J. Am. Chem. Soc. 94, 9241-9244 (1972).