Section 2. Structure and mechanism of non-heme iron-SAM dependent
3.7 Stereochemistry
3.7.3 Methoxylation
As mentioned above, Arg127 is electrostatically associated with Ppy (Figure 9), an interaction not seen in HMS. We considered the residue might exert the interaction to confine the enzyme reaction. Mutant R127L/D244E was first made and examined for its enzyme activities. The experiments showed the mutant retains the methylation activity (70%), indicating Arg127 has a modest effect on the Ppy binding. The active-site coordination in the structure of R127L/D244E is slightly dissimilar to that of D244E, where two water molecules are no longer sandwiched by D244E while -keto acid of Ppy remains chelation with the iron center (Figure 15a). To alter the bidentate binding mode, we re-introduced V300E into the R127L mutants. Two mutants R127L/V300E and R127L/D244A/V300E were made and subjected to enzyme examination. The results showed both mutants yield no β ePpy, while Ppy seemed consumed considerably. These two mutants were further crystallized to glean clues on this discrepancy (Figure 15b,c). To our surprise, a new chemical was identified in both structures, which was determined to be (2R,3R)--hydroxy--methoxy-phenylpropionic acid (Figure 15d). In the structures, Ppy adopts a new conformation with merely the carboxyl group engaging in the coordination. We reasoned Trp99 likely servers as a general base abstracting the pro-S proton from the benzylic carbon of Ppy to form an enol conformer. A water molecule assisted by Cys136 then attacks the benzylic carbon from Re-face and Trp99 reprotonates the -carbon to form a hydrated adduct. Cys136 further deprotonates the -hydroxyl group to attack the methylium from SAM to form the unusual ,-vicinal diasteromer (Figure 15e).
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4. Discussion
Having solved multiple complexed structures, we realized for the stereospecific C-MT reaction MppJ has evolved a non-heme iron center able to bind and orientate -ketoacid substrates and meanwhile developed a sandwiched bi-water device able to avoid formation of the reactive oxo-Fe(IV) species. In typical non-heme oxygenase, the iron center is commonly located at the hydrophobic center of the -barrel. In opposition to non-heme oxygenase, the iron center of MppJ is solvent exposed. Maybe we can create a hydrophobic environment near the active site of MppJ by introducing a long loop between 1 and 9. The engineered MppJ with long loop “cap” structure could have non-heme oxygenase activity, converting Ppy to mandelate or homogentisate. In addition, MppJ mutants were engineered for the first time able to perform hydration and methylation reactions through altering coordination chemistry for stereospecific new compounds. Glycopeptides and their derivatives are still the most attracting candidates in the development of new types of anti-MRSA or anti-VRE compounds. Biosynthetic engineering techniques, for example by addition of modified genes into producing strains, may also generate more powerful or equivalent glycopeptides but in a more diversified and economical fashion.
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Figure 1. Chemical structures of mannopeptimycins.
75
Figure 2. Multiple sequence alignments for MppJ and homologues.
Sequence alignments were performed by using ClustalW software (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The plot was generated by ESPript (http://espript.ibcp.fr/ESPript/ESPript). Secondary structure of MppJ was shown on the top of the sequence. Accession codes for MppJ (Q643C8), MmcR (Q9X5T6), LpOMT1 (Q9ZTU2), NcsB1 (Q84HC8), DnrK (Q06528), CalO1 (Q8KNE5), and RdmB (Q54527) are designated by the UniProtKB databank (http://www.uniprot.org/).
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a
b
77
c
Figure 3. Crystal structures of MppJ complexed with substrates or products.
(a) Overall structure of MppJ complexed with products SAH/MePpy. The structure of MppJ is a homo-dimer in the asymmetric unit. The bound ligands are well defined as shown in the electron density map. (b) Structure of MppJ-SAH-MePpy complex. 2Fo-Fc
electron density map of products SAH/MePpy contoured at 1 . (c) Structue of MppJ-SAM-Ppy complex. 2Fo-Fc electron density map of substrates SAM/Ppy contoured at 1
.
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Figure 4. Gel filtration and analytical ultracentrifugation analyses of MppJ.
(a) Size exclusive chromatography analysis of MppJ by using Superdex 75 10/300 column. (b) Size exclusive chromatography analysis of molecular weight standards in the same condition. The AUC data were analyzed by SedFit (http://www.analyticalultracentrifugation.com/default.htm). The calculated c(M) distribution (c) and c(S) distribution (d) as shown in the panels indicated MppJ forms a dimer. The insert grayscale bars indicate the residuals bitmap of each fit.
0
10000 30000 50000 70000 90000 110000 130000 150000 170000 190000 210000
c(M)
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Figure 5. Schematic topology of MppJ.
Topology diagram was generated by TopDraw program. The secondary structure of -helix and -sheet are shown in red and yellow, respectively. A subunit of MppJ is composed of two domains, which are N-terminal DD domain (residues 1-160) for protein dimerization and C-terminal MT catalytic domain (residues 161-337) for methyltransferation.
DD
MT
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a
b
Figure 6. Conformational change and SAM/SAH-binding.
(a) Superposition of binary (Ppy) and ternary (Ppy and SAM) structures revealed a protein conformational change (RMSD=0.968 for 344 C of MppJ). The structures of binary and ternary structures are shown in yellow and green, respectively. (b) Interactions between SAM and residues in MppJ structure. The figure was generated by LigPlot.
3.9 Å
81
a b
Figure 7. Ppy binding site and metal (Fe3+) coordination in MppJ structure.
(a) Ppy makes a distinctly asymmetric bidentate contact with ferric ion (Fe3+) through one of carboxylate oxygens and its adjacent -oxylate/keto oxygen. The 2 nitrogen of His243 and His295, the -ketoacid bidentate of Ppy, two water molecules sandwiched by the metal ion and the carboxylic group of Asp244 leading to a six-coordinate species.
(b) The phenyl portion of Ppy is located in a hydrophobic cavity formed by residues Phe287, Ile139, Met240, Phe287, Phe291, and Leu328.
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a
b
Figure 8. Metal ion determination by X-ray absorption spectroscopy (XAS) and electron paramagnetic resonance (EPR).
(a) The XAS spectrum of MppJ is shown in green. The spectra of standard Fe3+ and Fe2+ are colored in blue and red, respectively. (b) EPR analyses of MppJ. The metal is determined to be a high-spin ferric ion (S=5/2, g~4.3).
-300000
83
a b
c
Figure 9. Active site structure of MppJ and proposed catalytic mechanism.
(a) Active site arrangement of MppJ/SAM/Ppy complex. (b) Active site arrangement of MppJ/SAH/MePpy complex. The benzylic carbon of Ppy/MePpy forms hydrogen bonds with Trp99 and Cys136 from the si face and re face, respectively. The carboxylic group of Ppy/MePpy interacts with the guanidino group of Arg127. (c) The proposed methyltransferation mechanism of MppJ.
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MppJ-SAM (Kd=22.4±1.6 M) MppJ-Ppy (Kd=14.6±0.9 M) MppJ-SAH (Kd=2.5±0.4 M)
Figure 10. Isothermal titration calorimetry (ITC) analyses of MppJ.
ITC thermogram for MppJ binds with substrates (SAM and Ppy) or product (SAH).
Each exothermic heat pulse corresponds to injection of 2 l of ligands (1 mM) into the protein solution (0.1 mM); integrated heat areas constituted a differential binding curve that was fit to a standard single-site binding model (Origin 7.0, MicroCal iTC200).
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
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Figure 11. HPLC traces of enzymatic reactions for MppJ WT, D244L, and D244E mutants.
LC traces of enzymatic reactions for (a) WT MppJ, (b) D244L mutant, and (c) D244E mutant.
0 6 10 14 18 22 26 30 34 38
Time (min)
a b c
SAM SAH
Ppy MePpy
degration products
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a b
Figure 12. Active site structures of MppJ D244E and D244L mutants.
(a) Active site arrangement of MppJ D244E mutant. (b) Active site arrangement of MppJ D244L mutant, where dioxygen is side-on the iron center.
87
a
b
c
Figure 13. Chiral HPLC analyses of MppJ.
(a) Four enantiomers of -methylphenylalanine (MePhe) resolved by chiral HPLC. (b) Enzymatic products of MppJ and TyrB (aromatic amino acid-specific transaminase) at reaction courses 4 hr. (c) Enzymatic products of MppJ and TyrB at reaction courses 8 hr.
(2R,3S) (2S,3R)
(2R,3R)
(2S,3S)
(2S,3R)
(2S,3S)
(2S,3R)
(2S,3S)
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a b
c d
Figure 14. Ppy/4HPpy are covalently linked to Cys319.
(a) Structure of MppJ-SAH-MePpy complex. (b) Structure of MppJ-4HPpy complex. (c) 2Fo-Fc electron density map of covalently linked Ppy contoured at 1 . (d) 2Fo-Fc
electron density maps of 4HPpy in the active site and covalently linked 4HPpy contoured at 1 .
89
a b
c d
e
Figure 15. Active site structures of MppJ mutants and proposed mechanism for new compound.
(a) Active site structure of MppJ R127L/D244E mutant. (b) Active site structue of MppJ R127L/V300E mutant. (c) Active site structure of MppJ R127L/D244A/V300E mutant. (d) 2Fo-Fc electron density map of (2R,3R)--hydroxy--methoxy-phenylpropionic acid contoured at 1 . (e) The proposed hydration-methylation mechanism.
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Table 1. Data collection, phasing and refinement statistics for MppJ structures.
SeMppJ-SAH-MePPY MppJ-SAM-PPY MppJ-PPY MppJ-4HPPY
Data collection
Space group P212121 P212121 P212121 P212121
Cell dimensions
a, b, c (Å ) 64.3, 78.0, 138.2 57.5, 93.5, 142.2 57.4, 88.7, 137.0 59.5, 81.1, 137.4
(º) 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0
Peak Inflection Remote
Wavelength 0.97874 0.97893 0.96353 1.54 0.90 0.97
Resolution (Å ) 30.0-2.0(2.07-2.0) 30.0-2.0(2.07-2.0) 30.0-2.0(2.07-2.0) 19.0-2.45(2.54-2.45) 30.0-2.50(2.59-2.50) 30.0-2.40(2.49-2.40)
Rsym or Rmerge 11.0(72.3) 10.5(72.2) 11.0(75.1) 8.5(61.4) 12.2(62.0) 13.7(58.9)
I/I 23.3(3.25) 16.5(2.24) 16.3(2.29) 15.7(2.06) 13.3(2.67) 12.5(2.94)
Completeness (%) 99.9(100.0) 99.9(99.8) 99.9(99.8) 97.1(90.4) 99.1(98.6) 93.5(96.0)
Redundancy 10.3(10.2) 5.2(5.1) 5.1(5.1) 4.2(3.9) 4.9(5.0) 5.0(5.1)
Refinement
Resolution (Å ) 2.00 2.45 2.50 2.40
No. reflections 45227 27511 23457 23694
Rwork/ Rfree 0.201/0.256 0.171/0.238 0.174/0.248 0.172/0.248
No. atoms
*Highest resolution shell is shown in parenthesis.
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Resolution (Å ) 30.0-2.30(2.38-2.30) 30.0-2.50(2.59-2.50) 30.0-2.20(2.28-2.20) 30.0-2.10(2.18-2.10) 30.0-2.00(2.07-2.00)
Rsym or Rmerge 9.9(59.5) 9.5(66.9) 6.9(54.6) 8.7(52.1) 5.7(47.2)
I/I 16.6(3.0) 19.1(3.2) 25.0(3.3) 19.4(3.7) 26.6(3.5)
Completeness (%) 100.0(100.0) 99.9(100.0) 99.9(100.0) 99.9(100.0) 99.9(100.0)
Redundancy 4.9(4.9) 6.1(6.2) 6.1(6.2) 6.2(6.2) 5.8(5.3)
Refinement
Resolution (Å ) 2.30 2.50 2.20 2.10 2.00
No. reflections 30264 24097 34611 43121 48442
Rwork/ Rfree 0.165/0.227 0.182/0.255 0.162/0.229 0.147/0.196 0.155/0.211
No. atoms
*Highest resolution shell is shown in parenthesis.
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Table 2. Relative enzymatic activities (MT), enzyme colors and proposed residue functions.
Mutants Relative
activitya Color Function
M240L 0.88 green SAM/SAH binding
M241L 0.91 green SAM/SAH binding
M240L/M241L 0.70 green SAM/SAH binding
W99F 0.46 colorless Active site base
S104A 0.36 colorless Active site base
C136A 0.75 green SAM/SAH stabilization
C136H 0.01 green SAM/SAH stabilization
C319A 0.61 green Allosteric regulation
R127L 0.94 yellow Ppy binding
R127L/D244E 0.70 lime Ppy binding/water interaction
R127L/D244L 0.00 lime Ppy binding/water interaction
R127L/V300E 0.00 lime Hydrotase and methyltransferase
R127L/D244A/V300E 0.00 lime Hydrotase and methyltransferase
H243F 0.00 colorless Metal coordination
H295F 0.01 colorless Metal coordination
D244L 0.02 olive green Metal coordination
D244E 1.20 green Metal coordination
V300E 1.37 lime Metal coordination
D244A/V300E 0.26 lime Metal coordination
H243D/D244H 0.00 colorless Metal coordination
H243E/D244H 0.01 colorless Metal coordination
D244H/H295D 0.00 colorless Metal coordination
D244H/H295E 0.02 green Metal coordination
R127L/C136F/D244A/V300E 0.00 lime
Ppy binding/water activation/metal coordination/metal coordination
R127L/D244A/F291L/V300E 0.01 lime
Ppy binding/metal coordination/Ppy binding/metal
coordination R127L/C136F/D244A/F291L/V300E 0.00 lime
Ppy binding/water activation/metal coordination/Ppy
binding/metal coordination
a. The reactions of mutants were analyzed by HPLC; peak areas in triplicate were integrated and averaged; the reaction rates were calculated using linear regression equation;
relative activities were determined by dividing individual reaction rate with that of WT; the relative activity of WT is 1.0.
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References
1. Singh, M.P. et al. Mannopeptimycins, New Cyclic Glycopeptide Antibiotics Produced by Streptomyces hygroscopicus LL-AC98: Antibacterial and Mechanistic Activities. Antimicrob. Agents Chemother. 47, 62-69 (2003).
2. He, H. et al. Mannopeptimycins, Novel Antibacterial Glycopeptides from Streptomyces hygroscopicus, LL-AC98. J. Am. Chem. Soc. 124, 9729-9736 (2002).
3. Breukink, E. & de Kruijff, B. Lipid II as a target for antibiotics. Nat Rev Drug Discov 5, 321-323 (2006).
4. Koehn, F.E. New Strategies and Methods in the Discovery of Natural Product Anti-Infective Agents: The Mannopeptimycins. J. Med. Chem. 51, 2613-2617 (2008).
5. Malabarba, A. & Goldstein, B.P. Origin, structure, and activity in vitro and in vivo of dalbavancin. J. Antimicrob. Chemother. 55 Suppl 2, ii15-20 (2005).
6. Van Bambeke, F. Glycopeptides in clinical development: pharmacological profile and clinical perspectives. Curr Opin Pharmacol 4, 471-478 (2004).
7. Ruzin, A. et al. Mechanism of Action of the Mannopeptimycins, a Novel Class of Glycopeptide Antibiotics Active against Vancomycin-Resistant Gram-Positive Bacteria. Antimicrob. Agents Chemother. 48, 728-738 (2004).
8. Petersen, P.J., Wang, T.Z., Dushin, R.G. & Bradford, P.A. Comparative In Vitro Activities of AC98-6446, a Novel Semisynthetic Glycopeptide Derivative of the atural Product annopeptimycin α, and Other ntimicrobial gents against Gram-Positive Clinical Isolates. Antimicrob. Agents Chemother. 48, 739-746 (2004).
94
9. Haltli, B. et al. nvestigating β-Hydroxyenduracididine Formation in the Biosynthesis of the Mannopeptimycins. Chem. Biol. 12, 1163-1168 (2005).
10. Khare, M. & Keady, D. Antimicrobial therapy of methicillin resistant Staphylococcus aureus infection. Expert Opin Pharmacother 4, 165-177 (2003).
11. Magarvey, N.A., Haltli, B., He, M., Greenstein, M. & Hucul, J.A. Biosynthetic Pathway for Mannopeptimycins, Lipoglycopeptide Antibiotics Active against Drug-Resistant Gram-Positive Pathogens. Antimicrob. Agents Chemother. 50, 2167-2177 (2006).
12. Fontecave, M., Atta, M. & Mulliez, E. S-adenosylmethionine: nothing goes to waste. Trends Biochem. Sci. 29, 243-249 (2004).
13. Schubert, H.L., Blumenthal, R.M. & Cheng, X. Many paths to methyltransfer: a chronicle of convergence. Trends Biochem. Sci. 28, 329-335 (2003).
14. Martin, J.L. & McMillan, F.M. SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold. Curr. Opin. Struct. Biol.
12, 783-793 (2002).
15. Thibodeaux, C.J., Melançon, C.E. & Liu, H.-w. Natural-Product Sugar Biosynthesis and Enzymatic Glycodiversification. Angew. Chem. Int. Ed. 47, 9814-9859 (2008).
16. Thibodeaux, C.J., Melancon, C.E. & Liu, H.-w. Unusual sugar biosynthesis and natural product glycodiversification. Nature 446, 1008-1016 (2007).
17. Zubieta, C., He, X.-Z., Dixon, R.A. & Noel, J.P. Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferases. Nat Struct Mol Biol 8, 271-279 (2001).
18. Liu, Y.C. et al. Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds. Nat. Chem. Biol. 7, 304-309 (2011).
95
19. Chan, H.-C. et al. Regioselective deacetylation based on teicoplanin-complexed Orf2* crystal structures. Molecular BioSystems 7, 1224-1231 (2011).
20. Li, T.-L., Liu, Y.-C. & Lyu, S.-Y. Combining biocatalysis and chemoselective chemistries for glycopeptide antibiotics modification. Curr. Opin. Chem. Biol.
16, 170-178 (2012).
21. Houge-Frydrych, C.S., Gilpin, M.L., Skett, P.W. & Tyler, J.W. SB-203207 and SB-203208, two novel isoleucyl tRNA synthetase inhibitors from a Streptomyces sp. II. Structure determination. J. Antibiot. (Tokyo). 53, 364-372 (2000).
22. Huang, Y.-T. et al. In vitro Characterization of Enzymes Involved in the Synthesis of Nonproteinogenic Residue (2S,3S)-β-Methylphenylalanine in Glycopeptide Antibiotic Mannopeptimycin. ChemBioChem 10, 2480-2487 (2009).
23. Lin, H. S-Adenosylmethionine-dependent alkylation reactions: When are radical reactions used? Bioorg. Chem. 39, 161-170 (2011).
24. Akey, D.L. et al. A New Structural Form in the SAM/Metal-Dependent O‑
Methyltransferase Family: MycE from the Mycinamicin Biosynthetic Pathway.
J. Mol. Biol. 413, 438-450 (2011).
25. Gómez García, I. et al. The Crystal Structure of the Novobiocin Biosynthetic Enzyme NovP: The First Representative Structure for the TylF O-Methyltransferase Superfamily. J. Mol. Biol. 395, 390-407 (2010).
26. Kopycki, J.G. et al. Biochemical and Structural Analysis of Substrate Promiscuity in Plant Mg2+-Dependent O-Methyltransferases. J. Mol. Biol. 378, 154-164 (2008).
96
27. Duschene, K.S., Veneziano, S.E., Silver, S.C. & Broderick, J.B. Control of radical chemistry in the AdoMet radical enzymes. Curr. Opin. Chem. Biol. 13, 74-83 (2009).
28. Frey, P.A., Hegeman, A.D. & Ruzicka, F.J. The Radical SAM Superfamily. Crit.
Rev. Biochem. Mol. Biol. 43, 63-88 (2008).
29. Otwinowski, Z. & Minor, W. [20] Processing of X-ray diffraction data collected in oscillation mode, in Methods in Enzymology, Vol. Volume 276. (ed. Charles W. Carter, Jr.) 307-326 (Academic Press, 1997).
30. Kantardjieff, K.A. & Rupp, B. Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA, and protein–nucleic acid complex crystals. Protein Sci. 12, 1865-1871 (2003).
31. Ness, S.R., de Graaff, R.A.G., Abrahams, J.P. & Pannu, N.S. Crank: New methods for automated macromolecular crystal structure solution. Structure 12, 1753-1761 (2004).
32. De Graaff, R.A.G., Hilge, M., Van Der Plas, J.L. & Abrahams, J.P. Matrix methods for solving protein substructures of chlorine and sulfur from anomalous data. Acta Crystallographica Section D 57, 1857-1862 (2001).
33. Pannu, N.S. & Read, R.J. The application of multivariate statistical techniques improves single-wavelength anomalous diffraction phasing. Acta Crystallographica Section D 60, 22-27 (2004).
34. Abrahams, J.P. & Leslie, A.G.W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallographica Section D 52, 30-42 (1996).
35. Cowtan, K. Fitting molecular fragments into electron density. Acta Crystallographica Section D 64, 83-89 (2008).
97
36. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallographica Section D 62, 1002-1011 (2006).
37. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallographica Section D 66, 486-501 (2010).
38. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics.
Acta Crystallographica Section D 60, 2126-2132 (2004).
39. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of Macromolecular Structures by the Maximum-Likelihood Method. Acta Crystallographica Section D 53, 240-255 (1997).
40. Schuck, P. Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling. Biophys. J. 78, 1606-1619 (2000).
41. Ravel, B. & Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. Journal of Synchrotron Radiation 12, 537-541 (2005).
42. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658-674 (2007).
43. Dunwell, J.M., Purvis, A. & Khuri, S. Cupins: the most functionally diverse protein superfamily? Phytochemistry 65, 7-17 (2004).
44. Dunwell, J.M. Cupins: a new superfamily of functionally diverse proteins that include germins and plant storage proteins. Biotechnol. Genet. Eng. Rev. 15, 1-32 (1998).
45. Conrad, J.A. & Moran, G.R. The interaction of hydroxymandelate synthase with the 4-hydroxyphenylpyruvate dioxygenase inhibitor: NTBC. Inorg. Chim. Acta 361, 1197-1201 (2008).
98
46. Brownlee, J., He, P., Moran, G.R. & Harrison, D.H.T. Two Roads Diverged:
The Structure of Hydroxymandelate Synthase from Amycolatopsis orientalis in Complex with 4- ydroxymandelate†,‡. Biochemistry (Mosc). 47, 2002-2013 (2008).
47. Purpero, V. & Moran, G. The diverse and pervasive chemistries of the α-keto acid dependent enzymes. J Biol Inorg Chem 12, 587-601 (2007).
48. Moran, G.R. 4-Hydroxyphenylpyruvate dioxygenase. Arch. Biochem. Biophys.
433, 117-128 (2005).
49. Singh, S. et al. Structure and echanism of the ebeccamycin Sugar ′-O-Methyltransferase RebM. J. Biol. Chem. 283, 22628-22636 (2008).
50. Vidgren, J., Svensson, L.A. & Liljas, A. Crystal structure of catechol O-methyltransferase. Nature 368, 354-358 (1994).
51. Männistö, P.T. & Kaakkola, S. Catechol-O-methyltransferase (COMT):
Biochemistry, Molecular Biology, Pharmacology, and Clinical Efficacy of the New Selective COMT Inhibitors. Pharmacol. Rev. 51, 593-628 (1999).
52. Schluckebier, G., O'Gara, M., Saenger, W. & Cheng, X. Universal Catalytic Domain Structure of AdoMet-dependent Methyltransferases. J. Mol. Biol. 247, 16-20 (1995).
53. Costas, M., Mehn, M.P., Jensen, M.P. & Que, L. Dioxygen Activation at ononuclear onheme ron ctive Sites: En ymes, odels, and ntermediates.
Chem. Rev. 104, 939-986 (2004).
54. Solomon, E.I. et al. Geometric and Electronic Structure/Function Correlations in Non-Heme Iron Enzymes. Chem. Rev. 100, 235-350 (2000).
99
55. Decker, A. & Solomon, E.I. Dioxygen activation by copper, heme and non-heme iron enzymes: comparison of electronic structures and reactivities. Curr. Opin.
Chem. Biol. 9, 152-163 (2005).
56. Gerlt, J.A., Kreevoy, M.M., Cleland, W.W. & Frey, P.A. Understanding enzymic catalysis: the importance of short, strong hydrogen bonds. Chem. Biol.
4, 259-267 (1997).
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Appendix Curriculum Vitae
Personal Information Name: Yu-Chen Liu Date of Birth: 1979/09/20 Gender: male
Nationality: Taiwan
E-mail: ycl0920@gate.sinica.edu.tw Contact Number: +8862-27898070
Contact Address: 3L08, Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
Professional Profile
Current Research: Structural and functional studies of enzymes involved in the biosynthesis of natural products.
Affiliated Institution:
1. Genomics Research Center (GRC), Academia Sinica, Taipei, Taiwan.
2. Chemical Biology and Molecular Biophysics (CBMB) Program, Taiwan International Graduate Program (TIGP), Academia Sinica, Taipei, Taiwan.
3. Institute of Biochemical Sciences (IBS), National Taiwan University, Taipei, Taiwan.
Education and Experience
B.S., Department of Chemistry, National Cheng-Kung University, 1998- 2002.
M.S., Department of Biochemistry, National Cheng-Kung University Medical College, 2002- 2004.
R.A., Institute of Molecular Biology (IMB), Academia Sinica, 2004-2005.
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R.A., Genomics Research Center (GRC), Academia Sinica, 2007-2009.
Ph.D., Chemical Biology and Molecular Biophysics (CBMB) Program, Taiwan International Graduate Program (TIGP), Academia Sinica, 2009-2013.
Conferences and Workshops
1. Natural Products Conference 2012-Natural Products Synthesis and Biosynthesis, Puerto Calero, Lanzarote, Spain. (February 10-13, 2012)
2. 48th International Conference on Medicinal Chemistry-RICT 2012-Interfacing Chemical Biology and Drug Discovery, Poitiers, France. (July 4-6, 2012)
3. 1st OIST CCP4 school-From data processing to structure refinement and beyond, Okinawa, Japan. (December 5-9, 2011)
Academic Honors and Awards
1. Outstanding performance for the 2010 CBMB, IBC, and IBS retreat. (The second prize of poster and oral competition award)
2. Outstanding performance for the 2011 16th Biophysics Conference. (The first prize of poster and oral competition award)
3. Outstanding performance for the 2011 Symposium on Frontiers of Biomedical Sciences. (The first prize of poster and oral presentation award)-Prof. Jung-Yaw Lin Science and Education Foundation award.
4. Outstanding student research award, College of Life Science, National Taiwan University (2011).
5. Outstanding performance for the 2011 NSRRC 17th Users’ eeting & Workshop.
(The first prize of poster and oral presentation in biological science award-The Glory of Taiwan)
6. Outstanding student research award, 21st Wang Ming-Ning Memorial Foundation award (2011).
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7. Outstanding student research award, 1st GRC research staff award (2011).
8. TIGP student conference travel grant award. (The first prize of the grant award for the period of January-April 2012)
9. Outstanding student research award, 2nd GRC research staff award (2012).
Publications
1. Jia-Hau Shiu, Chiu-Yueh Chen, Long-Sen Chang, Yi-Chun Chen, Yen-Chin Chen, Yu-Hui Lo, Yu-Chen Liu, and Woei-Jer Chuang*, Solution Structure of -Bungarotoxin: The Functional Significance of Amino Acid Residues Flanking the RGD Motif in Integrin Binding, Proteins 57, 839-849 (2004).
2. Chiu-Yueh Chen, Jia-Hau Shiu, Yao-Husn Hsieh, Yu-Chen Liu, Yen-Chin Chen, Yi-Chun Chen, Wen-Yih Jeng, Ming-Jer Tang, Szecheng J. Lo, and Woei-Jer Chuang*, Effect of D to E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: Importance of the interactions between the D residue and integrin, Proteins 76, 808-821 (2009).
3. Yu-Ting Huang, Syue-Yi Lyu, Pei-Hsuan Chuang, Ning-Shian Hsu, Yi-Shan Li, Hsiu-Chien Chan, Chuen-Jiuan Huang, Yu-Chen Liu, Chang-Jer Wu, Wen-Bin Yang, and Tsung-Lin Li*, In vitro Characterization of Enzymes Involved in the Synthesis of Nonproteinogenic Residue (2S,3S)-β-Methylphenylalanine in Glycopeptide Antibiotic Mannopeptimycin, ChemBioChem 10, 2480-2487 (2009).
4. Hsiu-Chien Chan, Yu-Ting Huang, Syue-Yi Lyu, Chuen-Jiuan Huang, Yi-Shan Li, Yu-Chen Liu, Chia-Cheng Chou, Ming-Daw Tsai*, and Tsung-Lin Li*, Regioselective deacetylation based on teicoplanin-complexed Orf2* crystal structures, Mol. BioSyst.7, 1224-1231 (2011).
5. Yu-Chen Liu, Yi-Shan Li, Syue-Yi Lyu, Li-Jen Hsu, Yu-Hou Chen, Yu-Ting Huang, Hsiu-Chien Chan, Chuen-Jiuan Huang, Gan-Hong Chen, Chia-Cheng Chou,
Ming-xix
Daw Tsai, and Tsung-Lin Li*, Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds, Nat. Chem. Biol. 7, 304-309 (2011).
6. Tsung-Lin Li*, Yu-Chen Liu, and Syue-Yi Lyu, Combining biocatalysis and chemoselective chemistries for glycopeptide antibiotics modification, Curr. Opin.
Chem. Biol. 16, 170-178 (2012).
7. Hai-Chen Wu, Yi-Shan Li, Yu-Chen Liu, Syue-Yi Lyu, Chang-Jer Wu, and Tsung-Lin Li*, Chain elongation and cyclization in type III PKS DpgA, ChemBioChem 13, 862-871 (2012).
8. Xiao-Wei Zou†, Yu-Chen Liu†, Ning-Shian Hsu, Chuen-Jiuan Huang, Syue-Yi Lyu, Hsiu-Chien Chan, Chin-Yuan Chang, Hsien-Wei Yeh, Kuan-Hung Lin, Chang-Jer Wu, Ming-Daw Tsai, and Tsung-Lin Li*, Structure and mechanism of non-heme iron-SAM dependent methyltransferase and its engineering to hydratase.
(†These authors contribute equally) (submitted)
9. Syue-Yi Lyu, Yu-Chen Liu, Chuen-Jiuan Huang, Chin-Yuan Chang, Ning-Shian Hsu, Kuan-Hung Lin, Chang-Jer Wu, Ming-Daw Tsai, and Tsung-Lin Li*, Multi-phased acyltransferase crystal structures lead to discovery of new chemistry and chemicals. (submitted)
10. Chin-Yuan Chang, Syue-Yi Lyu, Yu-Chen Liu, Ning-Shian Hsu, Chih-Chung Wu, Cheng-Fong Tang, Kuan-Hung Lin, Jin-Yuan Ho, Chang-Jer Wu, Ming-Daw Tsai, and Tsung-Lin Li*, Unexpected dihydroxyarginine and hydroxycapreomycidine are the actual biosynthetic intermediacy toward streptolidine. (submitted) Patents
1. Structural and mechanistic basis for novel compound biosynthesis using the
1. Structural and mechanistic basis for novel compound biosynthesis using the