Chemistry & Biology
Structure of PimD-Desepoxypimaricin Complex
with a linear gradient (50%–90%) of methanol. The entire column cycle was as
following: 50% methanol, 0–3 min; increase methanol concentration up to
90%, 3–12 min, 90% methanol, 12–20 min; decrease methanol concentration
down to 50%, 20–25 min; 50% methanol, 25–30 min. Retention times for pimar-
icin and 4,5-desepoxypimaricin were 14.3 and 15.5 min, respectively.
Brautaset, T., Sletta, H., Nedal, A., Borgos, S.E., Degnes, K.F., Bakke, I.,
Volokhan, O., Sekurova, O.N., Treshalin, I.D., Mirchink, E.P., et al. (2008).
Improved antifungal polyene macrolides via engineering of the nystatin biosyn-
thetic genes in Streptomyces noursei. Chem. Biol. 15, 1198–1206.
Byrne, B., Carmody, M., Gibson, E., Rawlings, B., and Caffrey, P. (2003).
Biosynthesis of deoxyamphotericins and deoxyamphoteronolides by engi-
neered strains of Streptomyces nodosus. Chem. Biol. 10, 1215–1224.
Stopped-Flow Spectrophotometry
Rapid mixing experiments were conducted with a Hi-Tech Scientific instru-
ment (Bradford on Avon, UK) equipped with a photodiode array detector.
All the reaction kinetics were measured at 10ꢀC. Solutions of ferric PimD
(10 mM) in 100 mM KPi (pH 7.4) were mixed with buffered solutions of iodoso-
benzene (from 50 to 150 mM), or with 500 mM peroxynitrite dissolved in 10 mM
NaOH. Upon each mixing, total of 300 spectra were collected over various
timescales. Rate constants were estimated by globally fitting the kinetic
data at different ligand concentrations to the various models using singular
value decomposition analysis implemented in ProK software (Applied Photo-
physics, Leatherhead, UK). The kinetic constants obtained from the fitting
had uncertainties of %5%.
Caffrey, P., Aparicio, J.F., Malpartida, F., and Zotchev, S.B. (2008). Biosyn-
thetic engineering of polyene macrolides towards generation of improved
antifungal and antiparasitic agents. Curr. Top. Med. Chem. 8, 639–653.
Ceder, O., Hansson, B., and Rapp, U. (1977). Pimaricin. VIII. Structural and
configurational studies by electron impact and field desorption mass spec-
trometry, 13C (25.2 MHz) and 1H (270 MHz)-NMR spectroscopy. Tetrahedron
33, 2703–2714.
Chandrasena, R.E., Vatsis, K.P., Coon, M.J., Hollenberg, P.F., and Newcomb,
M. (2004). Hydroxylation by the hydroperoxy-iron species in cytochrome P450
enzymes. J. Am. Chem. Soc. 126, 115–126.
Cheron, M., Cybulska, B., Mazerski, J., Grzybowska, J., Czerwinski, A., and
Borowski, E. (1988). Quantitative structure-activity relationships in amphoter-
icin B derivatives. Biochem. Pharmacol. 37, 827–836.
ACCESSION NUMBERS
The atomic coordinates and structure factors (codes 2X9P and 2XBK) have
been deposited in the Protein Data Bank, Research Collaboratory for Struc-
tural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.
CCP4 (Collaborative Computational Project, Number 4). (1994). The CCP4
suite: programs for protein crystallography. Acta Crysallogr D 50, 760–763.
Cowtan, K. (2006). The Buccaneer software for automated model building. 1.
Tracing protein chains. Acta Crystallogr. D Biol. Crystallogr. 62, 1002–1011.
Cupp-Vickery, J.R., Han, O., Hutchinson, C.R., and Poulos, T.L. (1996).
Substrate-assisted catalysis in cytochrome P450 eryF. Nat. Struct. Biol. 3,
632–637.
ACKNOWLEDGMENTS
We thank Paul Ortiz de Montellano for valuable discussions, Potter Wickware
for critical reading of the manuscript, Chiung-Kuang Chen and the staff
members of beamline 8.3.1, James Holton, George Meigs and Jane Tanama-
chi, the Advanced Light Source at Lawrence Berkeley National Laboratory for
assistance with data collection. This work was supported by NIH RO1 grant
GM078553 (to L.M.P.) and Spanish Ministry of Science and Innovation grant
BIO2007-67585 (to J.F.A.). H.O. was supported by NIH RO1 grant GM25515
(to Paul R. Ortiz de Montellano). J.S.-A. was supported by F.P.U. fellowships
AP2005-3644 from the Ministry of Science and Education, Spain. The
Advanced Light Source is supported by the Director, Office of Science, Office
of Basic Energy Sciences, of the U.S. Department of Energy under Contract
No. DE-AC02-05CH11231. L.M.P. and H.O. designed research and analyzed
data; P.M.K. performed research; J.F.A. and J.S.-A. contributed new reagents,
L.M.P. wrote the manuscript.
DeLano, W.L. (2002). The PyMOL molecular graphics system (San Carlos, CA:
DeLano Scientific).
Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular
graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.
Ganis, P., Avitabile, G., Mechlinski, W., and Schaffner, C.P. (1971). Polyene
macrolide antibiotic amphotericin B. Crystal structure of the N-iodoacetyl
derivative. J. Am. Chem. Soc. 93, 4560–4564.
Gouet, P., Courcelle, E., Stuart, D.I., and Metoz, F. (1999). ESPript: multiple
sequence alignments in PostScript. Bioinformatics 15, 305–308.
Guengerich, F.P. (2003). Cytochrome P450 oxidations in the generation of
reactive electrophiles: epoxidation and related reactions. Arch. Biochem. Bio-
phys. 409, 59–71.
Harris, D.L., and Loew, G.H. (1998). Theoretical investigation of the proton
assisted pathway to formation of cytochrome P450 Compound I. J. Am.
Chem. Soc. 120, 8941–8948.
Received: March 19, 2010
Revised: May 12, 2010
Accepted: May 25, 2010
Published: August 26, 2010
Hirao, H., Kumar, D., and Shaik, S. (2006). On the identity and reactivity
patterns of the ‘‘second oxidant’’ of the T252A mutant of cytochrome
P450cam in the oxidation of 5-methylenenylcamphor. J. Inorg. Biochem.
100, 2054–2068.
REFERENCES
Aparicio, J.F., Caffrey, P., Gil, J.A., and Zotchev, S.B. (2003). Polyene antibiotic
Holton, J., and Alber, T. (2004). Automated protein crystal structure determina-
biosynthesis gene clusters. Appl. Microbiol. Biotechnol. 61, 179–188.
tion using ELVES. Proc. Natl. Acad. Sci. USA 101, 1537–1542.
Aparicio, J.F., Mendes, M.V., Anton, N., Recio, E., and Martin, J.F. (2004).
Imai, M., Shimada, H., Watanabe, Y., Matsushima-Hibiya, Y., Makino, R.,
Koga, H., Horiuchi, T., and Ishimura, Y. (1989). Uncoupling of the cytochrome
P-450cam monooxygenase reaction by a single mutation, threonine-252 to
alanine or valine: possible role of the hydroxy amino acid in oxygen activation.
Proc. Natl. Acad. Sci. USA 86, 7823–7827.
Polyene macrolide antibiotic biosynthesis. Curr. Med. Chem. 11, 1645–1656.
Bach, R.D., and Dmitrenko, O. (2004). Strain energy of small ring hydrocar-
bons. Influence of C-H bond dissociation energies. J. Am. Chem. Soc. 126,
4444–4452.
Jin, S., Makris, T.M., Bryson, T.A., Sligar, S.G., and Dawson, J.H. (2003). Epox-
idation of olefins by hydroperoxo-ferric cytochrome P450. J. Am. Chem. Soc.
125, 3406–3407.
Baginski, M., Sternal, K., Czub, J., and Borowski, E. (2005). Molecular model-
ling of membrane activity of amphotericin B, a polyene macrolide antifungal
antibiotic. Acta Biochim. Pol. 52, 655–658.
Jin, S., Bryson, T.A., and Dawson, J.H. (2004). Hydroperoxoferric heme
intermediate as a second electrophilic oxidant in cytochrome P450-catalyzed
reactions. J. Biol. Inorg. Chem. 9, 644–653.
Baginski, M., Czub, J., and Sternal, K. (2006). Interaction of amphotericin B
and its selected derivatives with membranes: molecular modeling studies.
Chem. Rec. 6, 320–332.
Brajtburg, J., Powderly, W.G., Kobayashi, G.S., and Medoff, G. (1990). Am-
photericin B: current understanding of mechanisms of action. Antimicrob.
Agents Chemother. 34, 183–188.
Kellner, D.G., Hung, S.C., Weiss, K.E., and Sligar, S.G. (2002). Kinetic charac-
terization of compound I formation in the thermostable cytochrome P450
CYP119. J. Biol. Chem. 277, 9641–9644.
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