purpuratus.
Puneet Juneja1, Ashit Rao2, Helmut Cölfen2, Kay Diederichs1, Wolfram Welte1
1Department of Biology, 2Department of Chemistry Universitätsstraße 10, D-78457 Konstanz, Germany
Published in Acta Cryst F
Acta Crystallogr F Struct Biol Commun. 2014 Feb 1;70 (Pt 2):260-2. doi:
10.1107/S2053230X14000880.
7.1 Abstract
Sea urchin spicules have a calcitic mesocrystalline architecture closely associated with the matrix of proteins and amorphous mineral. The mechanism underlying spicule formation involves complex processes encompassing spatio-temporally regulated organic-inorganic interactions. C-type lectin domains are present in several spicule matrix proteins in Strongylocentrotus purpuratus implying their role in spiculogenesis. In this study, the C-type lectin domain of SM50 was over-expressed, purified and crystallized using a vapour-diffusion method. The crystal diffracted to a resolution of 2.85 Å and belongs to space group P212121, with unit-cell parameters a = 100.6, b = 115.4, c = 130.6 Å, α = β = γ = 90°. Assuming 50% solvent content, we expect six chains in the asymmetric unit.
Key words: C-type lectin; SM50; spiculogenesis; Strongylocentrotus purpuratus
7.2 Introduction
The phenomenon of biomineralization encompasses diverse and widespread processes that involve organic-inorganic interactions by which organisms form composite, hierarchical material (147–149). The sea urchin spicule is a model system for investigating the mechanisms of calcium carbonate biomineralization (150). The spicule has a mesocrystalline architecture composed of crystallographically aligned calcite particles (50-200 nm) organized in a matrix of proteins and amorphous calcium carbonate (151). On account of this structure, the spicule has unique properties such as a single crystal-like diffraction and a conchoidal fracture surface typical of amorphous materials.
Hence the mechanisms underlying spicule formation have attracted interest from multiple disciplines (28, 152, 153).
Among the proteins regulating spicule formation, SM50 is a 48.5 kDa non-glycosylated, secreted protein with an alkaline pI (154, 155). The N-terminal region of SM50 harbors a C-type lectin (CTL) domain (13.6 kDa), which can affect calcium carbonate mineralization (28, 156). Although their functions are yet unknown, CTL domains are also present in other proteins associated with the sea urchin spicule such as the SM30 family (157). Thus CTL domains appear to play an important role in calcium carbonate biomineralization. In nature, proteins with the CTL fold such as the Type II antifreeze proteins, phospholipase receptors and coagulation factor binding proteins carry diverse functions (158). These proteins can also bind to carbohydrate ligands, Ca2+ ions and form oligomers (27). To elucidate their role in biomineralization, the structural and functional understanding of the CTL domain with respect to calcium carbonate mineralization is important. The highest sequence identity of the CTL domain from Strongylocentrotus purpuratus with a protein of known structure is 32 % (snake type CTL, PDB ID: 3UBU).
The snake CTL binds specifically to blood platelet glycoproteins and inhibits adhesion and aggregation (159). Here we report the purification, crystallization and preliminary X-ray analysis of the CTL domain of SM50 spicule matrix protein in fusion with SUMO protein.
7.3 Experimental Procedures Overexpression and Purification
Protein expression and purification was carried out as described previously (Rao et al., 2013). Briefly the CTL domain (13.6 kDa) of larval spicule matrix protein, SM50 from Strongylocentrotus purpuratus, N-terminally fused with a cleavable (Ulp1 protease) His6 -SUMO tag was cloned in pET24a vector (Fig.1). The Escherichia coli BL21 CodonPlusRIL were transformed with the pET24a vector and cultured in LB medium at 30°C. On reaching an OD600 of 0.6, expression was induced with 0.5 mM IPTG overnight at 20°C. Cells were harvested and suspended in lysis buffer, 20 mM HEPES, 50 mM NaCl, 10 mM β-mercaptoethanol pH 7.0, pepstatin (10 pg/ml), 0.5 mM PMSF and lysozyme (Roth, Germany) (0.5 mg/ml) and incubated for 30 minutes on ice. The resulting cell lysate was centrifuged at 16,000 rpm for 30 minutes and resulting supernatant was passed over a Ni-NTA column (Qiagen). After initial washing with 5 bed volumes of 20 mM HEPES, 50 mM NaCl, 0.5 mM imidazole, 10 mM β-ME, pH 7.0, the protein was eluted with 20 mM HEPES, 50 mM NaCl, 200 mM imidazole, 10mM β-ME, pH 7.0. The eluted protein was concentrated with the Vivaspin concentrators (5kDa cutoff) and loaded on a Superdex 75 column (GE Healthcare) for final purification in 20 mM HEPES, 50 mM NaCl, 10 mM β-ME, pH 7.0. A single peak was observed and the peak fractions were analyzed with SDS Gel electrophoresis (Fig.2). When the SUMO tag was cleaved, the CTL domain was unstable and aggregated, therefore crystallization was done with the SUMO-CTL fusion protein. HEPES pH 7.5 and 70% MPD) using a protein to reservoir ratio of 1 (v/v) (0.2 µl protein, 0.2 µl reservoir) at 18 °C. Further optimization was done in both hanging drop and sitting drop.
Data collection and processing
The harvested crystals were directly flash-frozen in liquid nitrogen. Data were collected at X06DA beam line, Swiss Light source (SLS), Villigen, Switzerland. The best crystal diffracted isotropically to a resolution of 2.85 Å. The diffraction data set was processed with the X-ray Detector Software (XDS Program Package) (160, 161). The space group assignment was done with Pointless (162) and further analysis of data quality was carried out with Phenix.Xtriage (163).
7.4 Results and Discussion
The SUMO-CTL crystals appeared after three weeks. The crystals were very fragile.
When wells were opened, crystals tended to lose their crystalline shape and changed to spherical droplets, resembling droplets observed during phase separation of organic solvents such as PEG and MPD. Addition of glycerol and ethylene glycol did not improve the crystals quality. Crystals were optimized in hanging and sitting drop conditions, the best diffracting crystals were obtained using the sitting drop method (Fig.3). Crystals were quickly harvested using a CryoLoop™ (Hampton research) directly for flash freezing in liquid nitrogen. The best crystal diffracted to a resolution of 2.85 Å and belongs to the orthorhombic space group P212121 (Table 1). The SUMO-CTL fusion protein with total molecular weight of 25.9 kDa is monomeric in solution as shown by analytical ultracentrifugation (28). Further analysis with Phenix.Xtriage based on sequence composition gave a solvent content of 50.2% and a Matthews’s coefficient of 2.47 Å3 Da-1 (164), with six chains in the asymmetric unit. Attempts to solve the structure with molecular replacement models, SUMO protein (PDB:ID 3QHT, 3PGE, 3TIX), snake CTL (PDB:ID 3UBU) and combination of both were unsuccessful.
Therefore, presently we are working on the crystallization of Se-Met labeled SUMO-CTL protein.
Acknowledgements
AR acknowledges Prof. Dr. Martin Scheffner for his support and encouragement. We would like to acknowledge Konstanz Research School Chemical Biology for financial assistance and staff at Swiss Light Source (Villigen, Switzerland) for their support during data collection.
Table 1.
Diffraction data statistics
Diffraction source X06DA
Wavelength (Å) 1.000
Rotation range per image (°) 0.1
Total rotation range (°) 180
Exposure time per image (s) 0.1
Space group P212121
Unit-cell parameters (Å,) a = 100.6, b =115.4, c =130.6,
Mosaicity (°) 0.228
Resolution range (Å) 2.85 (3.02-2.85)
Total No. of reflections 242290 (35688) No. of unique reflections 36606 (5688)
Completeness (%) 99.5 (97.5)
Multiplicity 6.6 (6.2)
Mean I/ (I) 6.38 (1.07)
CC (1/2)* (%) 98.4 (42.7)
Rmerge (%) 29 (166.1)
*(165)
Values in parentheses are for outer shell
FIGURES
FIGURE 1. Sequence of SUMO-CTL fusion protein used for crystallization. SUMO domain with N-terminal His6-tag is highlighted in green and CLT domain of SM50 protein (NCBI Reference Sequence: NP_999775.1) in magenta.
FIGURE 2. SDS PAGE showing purified SUMO-CTL after size exclusion chromatography purification (Superdex 75). Individual molecular weight of SUMO and CTL domain are 12.1 kDa and 13.6 kDa respectively. The SUMO-CTL runs at an apparent molecular weight near 25 kDa.
FIGURE 3. Crystals of SUMO-CTL fusion protein grown in sitting drop vapour diffusion method with protein to reservoir ratio of 1:1, in 0.1 mM HEPES pH 7.5, 70%
MPD. Crystals grew up to a length of 150 µm.
Final Discussion
X-ray crystallography is a fundamental tool for understanding molecular structure and functional mechanisms of the proteins. However, protein crystallization is a painstaking, frustrating and time consuming process. To make well-diffracting protein crystals one has to go through a long process of designing protein constructs with enhanced crystallization propensity or to find a suitable protein homologue that could be crystallized more easily.
As it is hard to say in advance which construct or homologue will crystallize at the end, one ends up in crystallizing different constructs and proteins in parallel. There are some general rules laid down based on already known structures and success stories but every protein has its own story.
I started my work with crystallization of eukaryotic Cysteine loop receptors (CLRs). At the time I started there was no structure available for any eukaryotic CLRs. CLRs are homo-or-hetero pentameric membrane receptors, which perform manifold functions in the neuronal systems.
The first receptor that I worked on was the nicotinic acetylcholine receptor (nAChR) from Torpedo californica. It is a hetero-pentameric receptor with four different subunits and a homologue of the mammalian receptor found in the neuromuscular junctions.
Because of its high natural expression in the electric organ of Torpedo californica, it was chosen for crystallization. Several labs had tried to crystallize this nAChR for the last 15 years but failed. However a new strategy for crystallization of Torpedo californica nAChR was adopted. I devised an affinity chromatography based on its ligand alpha-bungarotoxin, allowing to purify α-bunagrotoxin-nAChR complex from membranes. The stability of alpha-bungarotoxin-nAChR complex was proven in different detergents and the protein dimeric native state was preserved. Nevertheless, crystallization attempts in various detergents were unsuccessful. The approach to crystallize a complex of the alpha-bungarotoxin-nAChR with DARPins failed completely as no high affinity binding DARPins could be selected.
Another member of the CLR family chosen for crystallization was the Gamma amino butyric acid (GABAA) β3 receptor from Rattus norvegicus. Our collaborative partners provided the expressed protein. The GABAA β3 receptor was purified from Sf9 cells and tested for stability in different detergents, at different salt concentrations, various pH
values and in presence of ligands and lipids. The GABAA β3 receptor was found to be unstable and polydisperse under all conditions. Nevertheless, crystallization attempts were made but without any success. In collaboration with our partners in Heidelberg we tried to establish an expression system for Alpha7 nAChR from Rattus norvegicus in Drosophila melanogaster eyes but without success.
The focus was shifted toward finding more stable CLR for crystallization and I started looking into gene sequences of organisms living in hot environments. The only available genome of thermophilic eukaryotes, the fungus Chaetomium thermophilum did not contain any CLR gene. Another thermophilic eukaryote was an annelid Alvinella pompejana whose EST sequence database was available. I could put the EST sequences together and retrieved two full-length open reading frames of two CLRs, which were named Alv-a1-pHCl and Alv-a9. Alv-a1-pHCl shares 36 % sequence identity with human Glycine receptor and Alv-a9 had 27 % sequence identity with human Alpha 9 receptor.
One of these, Alv-a1-pHCl, could be expressed as a functional ligand-gated channel in Xenopus oocytes. It opens transiently at an acidic pH of 3 and was permeable to chloride ions. Alv-a1-pHCl was further characterized in detail and was found to be sensitive to picrotoxin. An Sf9 expression system was established and four different constructs tAlv-a1-pHCl, tAlv-a1-pHCl-AGT, thAlv-tAlv-a1-pHCl, thAlv-a1-pHCl-AGT could be expressed.
Construct tAlv-a1-pHCl was chosen for large-scale expression and purification. It was found that tAlv-a1-pHCl was stable up to 65 °C. Its temperature stability was 15-20 °C higher than that of the Torpedo nAChR. Being a thermostable protein, it is a good candidate for crystallization.
In another project, I worked on the crystallization and structure analysis of Chrorismatases. Chorismatase are involved in degradation of chorismate to pyruvate and benzoic acid derivatives. Chorismate is a central branching point for many biosynthetic pathways in plants, fungi and bacteria and thus are of biotechnological importance. Two different chorismatase homologues, FkbO and Hyg5 from Streptomyces hygroscopicus, were successfully crystallized and the structure was solved at 1Å and 1.9Å resolution, respectively. Our FkbO structure was the first determined of a chorismatase and allowed us to propose a universal functional enzymatic mechanism for chorismate hydrolysis. The Hyg5 molecular mechanism is still under investigation.
In a further project, the C-type like Lectin (CTL) domain of the SM50 protein from Strongylocentrotus purpuratus was investigated which is known to be important for calcium carbonate mineralization. It was purified in complex with SUMO and crystallized. Crystals diffracted to a resolution of 2.85 Å. The crystal structure analysis of CTL is in progress.
Record of Observation
The work in the thesis was performed in collaboration with other colleagues. In the following, I list my contribution.
Chapter 1: An internally modulated, thermostable, pH sensitive Cys-loop receptor from the hydrothermal vent worm Alvinella pompejana
Puneet Juneja, Reinhold Horlacher, Daniel Bertrand, Ryoko Krause, Fabrice Marger, Wolfram Welte
Chapter 2: Stability of Alpha-Bungarotoxin affinity purified Torpedo nicotinic acetylcholine receptor in lipid based detergents.
Chapter 3: Expression, Purification and Crystallization of GABAA β3 receptor from Rattus norvegicus.
My contributions, - Protein purification - Crystallization
Chapter 4: Expression and purification of Rattus norvegicus Alpha 7 nicotinic acetylcholine receptor expressed in Drosophila melanogaster photoreceptor cells.
My contributions, - Protein expression - Protein purification
Chapter 5: Mechanistic implications for the chorismatase FkbO based on the crystal
structure
Puneet Juneja, Florian Hubrich, Kay Diederichs, Wolfram Welte, Jennifer N. Andexer J Mol Biol. 2014
My contributions, - Protein purification - Crystallization - Data collection - Structure solution - Drafted the manuscript
Chapter 6: Crystal structure and mechanism of Hyg5 type chorismatase.
Puneet Juneja, Florian Hubrich, Kay Diederichs, Wolfram Welte, Jennifer N. Andexer Manuscript in Preparation
My contributions, - Protein purification - Crystallization - Data collection - Structure solution - Drafted the manuscript
Chapter 7: Crystallization and preliminary X-ray analysis of the C-type lectin domain of the spicule matrix protein SM50 from Strongylocentrotus purpuratus.
Puneet Juneja, Ashit Rao, Helmut Cölfen, Kay Diederichs, Wolfram Welte Acta Crystallogr F Struct Biol Commun. 2014
My contributions, - Protein purification - Crystallization - Data collection
- Drafted the manuscript
Miscellaneous
Chapter 5: Mechanistic implications for the chorismatase FkbO based on the crystal structure
PDB ID 4bps
Protein: Chorismatase- FkbO
Protein Modification: Selenomethionine labeled
Data Collection date: 2012 (somehow data has been shifted to 5-February-folder on disk).
Data on Disk: nfs/loop1/synchrotron/SLS-2013/feb05/pj/504-A2_1_1????? .cbf
Chapter 6: Crystal structure and mechanism of Hyg5 type chorismatase.
PDB ID To be submitted Protein: Chorismatase- Hyg5
Protein Modification: No modification Data Collection date: 12-September -2013
Data on Disk: nfs/loop1/synchrotron/SLS-2013/sep12/pj/hi5-A1_4_1????? .cbf
Chapter 7: Crystallization and preliminary X-ray analysis of the C-type lectin domain of the spicule matrix protein SM50 from Strongylocentrotus purpuratus.
PDB ID not applicable
Protein: CTL domain of SM 50 protein with N-terminal SUMO fusion.
Protein Modification: No modification Data Collection date: 06-March-2013
Data on Disk: nfs/loop1/synchrotron/SLS-2013/mar06/pj/cl-pj1_2_????? .cbf
REFERENCES Epilepsies Associated with GABAA Receptor Subunit Mutations. Epilepsy Curr.
9, 18–23
4. Hoda, J.-‐C., Gu, W., Friedli, M., Phillips, H. A., Bertrand, S., Antonarakis, S. E., Goudie, D., Roberts, R., Scheffer, I. E., Marini, C., Patel, J., Berkovic, S. F., Mulley, J. C., Steinlein, O. K., and Bertrand, D. (2008) Human nocturnal frontal lobe epilepsy: pharmocogenomic profiles of pathogenic nicotinic acetylcholine receptor beta-‐subunit mutations outside the ion channel pore. Mol. expression profile of the GMR-‐GAL4 driver in Drosophila melanogaster. Genet.
Mol. Res. GMR 11, 1997–2002
8. Tzin, V., and Galili, G. (2010) New Insights into the Shikimate and Aromatic Amino Acids Biosynthesis Pathways in Plants. Mol. Plant 3, 956 –972
9. Dosselaere, F., and Vanderleyden, J. (2001) A Metabolic Node in Action:
Chorismate-‐Utilizing Enzymes in Microorganisms. Crit. Rev. Microbiol. 27, 75–
131 chorismate-‐utilising enzymes by 2-‐amino-‐4-‐carboxypyridine and 4-‐
carboxypyridone and 5-‐carboxypyridone analogues. Org Biomol Chem 8, 3534–3542
14. Pollegioni, L., Schonbrunn, E., and Siehl, D. (2011) Molecular basis of glyphosate glycosite-‐specific, cancer-‐related carbohydrate structures in trypsin-‐digested human plasma. Anal. Biochem. 408, 71–85 expressed on peritoneal phagocytic cells with an immature dendritic cell-‐like phenotype is involved in uptake of oligomannose-‐coated liposomes and Interactions Preserve Angiogenesis in Anti-‐VEGF Refractory Tumors. Cell 156, 744–758
Distinct Ligands for the C-‐type Lectin Receptors Mincle and Dectin-‐2 in the Phylogeny.fr: robust phylogenetic analysis for the non-‐specialist. Nucleic Acids Res. 36, W465–469
40. Heuberger, E. H. M. L., Veenhoff, L. M., Duurkens, R. H., Friesen, R. H. E., and Poolman, B. (2002) Oligomeric state of membrane transport proteins
analyzed with blue native electrophoresis and analytical ultracentrifugation. J. transmembrane helices reveals widespread rearrangements during opening of P2X receptor channels. Neuron 54, 263–274
43. Krause, R. M., Buisson, B., Bertrand, S., Corringer, P. J., Galzi, J. L., Changeux, J. P., and Bertrand, D. (1998) Ivermectin: a positive allosteric effector of the alpha7 neuronal nicotinic acetylcholine receptor. Mol. Pharmacol. 53, 283–294
44. Hurst, R. S., Hajós, M., Raggenbass, M., Wall, T. M., Higdon, N. R., Lawson, J. A.,
receptors by benzylidene anabaseines and nicotine. J. Pharmacol. Exp. Ther. parasitic nematode Haemonchus contortus. Mol. Pharmacol. 75, 1347–1355 58. Mounsey, K. E., Dent, J. A., Holt, D. C., McCarthy, J., Currie, B. J., and Walton, S. F.
(2007) Molecular characterisation of a pH-‐gated chloride channel from Sarcoptes scabiei. Invertebr. Neurosci. IN 7, 149–156
59. Brownlee, D. J., Holden-‐Dye, L., and Walker, R. J. (1997) Actions of the anthelmintic ivermectin on the pharyngeal muscle of the parasitic nematode, Ascaris suum. Parasitology 115 ( Pt 5), 553–561
60. Wolstenholme, A. J., and Rogers, A. T. (2005) Glutamate-‐gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics.
Parasitology 131 Suppl, S85–95 comparison of cuticle and interstitial collagens from annelids living in shallow sea-‐water and at deep-‐sea hydrothermal vents. J. Mol. Biol. 246, 284–294
69. Holder, T., Basquin, C., Ebert, J., Randel, N., Jollivet, D., Conti, E., Jékely, G., and the ligand-‐binding domain of nicotinic receptors. Nature 411, 269–276
80. Bocquet, N., Nury, H., Baaden, M., Le Poupon, C., Changeux, J.-‐P., Delarue, M., and Corringer, P.-‐J. (2009) X-‐ray structure of a pentameric ligand-‐gated ion channel in an apparently open conformation. Nature 457, 111–114
81. Hilf, R. J. C., and Dutzler, R. (2009) Structure of a potentially open state of a proton-‐activated pentameric ligand-‐gated ion channel. Nature 457, 115–118 82. Hamilton, S. L., McLaughlin, M., and Karlin, A. (1977) Disulfide bond cross-‐ Effects of lipid-‐analog detergent solubilization on the functionality and lipidic
cubic phase mobility of the Torpedo californica nicotinic acetylcholine receptor. J. Membr. Biol. 243, 47–58
86. Jones, O. T., Eubanks, J. H., Earnest, J. P., and McNamee, M. G. (1988) A minimum number of lipids are required to support the functional properties of the nicotinic acetylcholine receptor. Biochemistry (Mosc.) 27, 3733–3742
87. Hamouda, A. K., Sanghvi, M., Sauls, D., Machu, T. K., and Blanton, M. P. (2006) Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor. Biochemistry (Mosc.) 45, 4327–4337
88. Welte, W., Leonhard, M., Diederichs, K., Weltzien, H.-‐U., Restall, C., Hall, C., and Chapman, D. (1989) Stabilization of detergent-‐solubilized Ca2+-‐ATPase by poly(ethylene glycol). Biochim. Biophys. Acta BBA -‐ Biomembr. 984, 193–199 89. Bhushan, A., and McNamee, M. G. (1990) Differential scanning calorimetry and
Fourier transform infrared analysis of lipid-‐protein interactions involving the nicotinic acetylcholine receptor. Biochim. Biophys. Acta 1027, 93–101
90. Hovers, J., Potschies, M., Polidori, A., Pucci, B., Raynal, S., Bonneté, F., Serrano-‐
Vega, M. J., Tate, C. G., Picot, D., Pierre, Y., Popot, J.-‐L., Nehmé, R., Bidet, M., Mus-‐
Veteau, I., Busskamp, H., Jung, K.-‐H., Marx, A., Timmins, P. A., and Welte, W.
(2011) A class of mild surfactants that keep integral membrane proteins water-‐soluble for functional studies and crystallization. Mol. Membr. Biol. 28, 171–181
91. Dainese, E., Oddi, S., and Maccarrone, M. (2008) Lipid-‐mediated dimerization of beta2-‐adrenergic receptor reveals important clues for cannabinoid receptors.
Cell. Mol. Life Sci. CMLS 65, 2277–2279
92. Santiago, J., Guzmán, G. R., Rojas, L. V., Marti, R., Asmar-‐Rovira, G. A., Santana, L.
F., McNamee, M., and Lasalde-‐Dominicci, J. A. (2001) Probing the Effects of Membrane Cholesterol in the Torpedo californica Acetylcholine Receptor and the Novel Lipid-‐exposed Mutation αC418W in XenopusOocytes. J. Biol. Chem. Cardiopulmonary arrest and resuscitation disrupts cholinergic anti-‐
inflammatory processes: a role for cholinergic α7 nicotinic receptors. J.
Neurosci. Off. J. Soc. Neurosci. 31, 3446–3452
98. Castro, N. G., and Albuquerque, E. X. (1995) alpha-‐Bungarotoxin-‐sensitive hippocampal nicotinic receptor channel has a high calcium permeability.
Biophys. J. 68, 516–524
99. Orr-‐Urtreger, A., Göldner, F. M., Saeki, M., Lorenzo, I., Goldberg, L., De Biasi, M., Dani, J. A., Patrick, J. W., and Beaudet, A. L. (1997) Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-‐bungarotoxin binding sites and hippocampal fast nicotinic currents. J. Neurosci. Off. J. Soc. Neurosci. Bacterial production of trans-‐dihydroxycyclohexadiene carboxylates by metabolic pathway engineering. Microbiology 142, 1005–1012
105. Sprenger, G. A. (2007) From scratch to value: engineering Escherichia coli wild type cells to the production of L-‐phenylalanine and other fine chemicals derived from chorismate. Appl Microbiol Biotechnol 75, 739–749
106. Andexer, J. N., Kendrew, S. G., Nur-‐e-‐Alam, M., Lazos, O., Foster, T. A., bifunctional chorismatase that links the shikimate pathway to ubiquinone and xanthomonadins biosynthetic pathways. Mol Microbiol 87, 80–93
108. He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) Conservation of Mechanism in Three Chorismate-‐Utilizing Enzymes. J Am Chem Soc 126, 2378–2385
109. Kerbarh, O., Ciulli, A., Howard, N. I., and Abell, C. (2005) Salicylate Biosynthesis: Overexpression, Purification, and Characterization of Irp9, a Bifunctional Salicylate Synthase from Yersinia enterocolitica. J. Bacteriol. 187, 5061 – 5066
110. Chook, Y. M., Gray, J. V., Ke, H., and Lipscomb, W. N. (1994) The Monofunctional Chorismate Mutase from Bacillus subtilis: Structure
Determination of Chorismate Mutase and Its Complexes with a Transition Enzymes. Biochemistry (Mosc.) 50, 7476–7483
113. Wu, K., Chung, L., Revill, W. P., Katz, L., and Reeves, C. D. (2000) The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene cluster isolated from Streptomyces hygroscopicus ATCC 29253, a rapamycin-‐
113. Wu, K., Chung, L., Revill, W. P., Katz, L., and Reeves, C. D. (2000) The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene cluster isolated from Streptomyces hygroscopicus ATCC 29253, a rapamycin-‐