Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone

143 Pages·2009·3.17 MB·English

Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone

By

Xin Lin

B.S., University of Science & Technology of China, 2002

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
IN THE DEPARTMENT OF CHEMISTRY AT BROWN UNIVERSITY

Providence, Rhode Island
May, 2009
© Copyright 2009 by Xin Lin
This dissertation by Xin Lin is accepted in it present form
by the department of Chemistry as satisfying the
dissertation requirement for the degree of Doctor of Philosophy

Date __________________ _________________________
David E. Cane

Recommended to the Graduate Council

Date __________________ _________________________

Amit Basu

Date __________________ _________________________

Jason K. Sello

Approved by the Graduate Council

Date __________________ _________________________
Dean of the graduate school, Shelia Bonde

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VITA

Xin Lin was born on October 01, 1979, in Fuzhou, China. She went to University of
Science & Technology of China for undergraduate study since 1997 and graduated with a
B.S. in Chemistry in 2002. Xin joined Brown University in August, 2002 and worked in
Prof. David E. Cane’s research group for her Ph.D. in Chemistry.

Publications:

1. Lin, Xin; Cane, David E. “Mechanism of Epi-isozizaene Synthase-Catalyzed
Cyclization” Manuscript in preparation.

2. Zhao, Bin; Lin, Xin; Lei, Li; Lamb, David C.; Kelly, Steven L.; Waterman, Michael
R.; Cane, David E. “Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone in
Streptomyces Coelicolor A3 (2)” J. Biol. Chem. 2008, 283, 8183 - 8189.

3. Liu, Tiangang; Lin, Xin; Zhou, Xiufen; Deng, Zixin; Cane, David E. “Mechanism of
Thioesterase-Catalyzed Chain Release in the Biosynthesis of the Polyether Antibiotic
Nanchangmycin” Chemistry & Biology, 2008, 15, 449 – 458

4. Lin, Xin; Hopson, Russell; Cane, David E. “Genome Mining in Streptomyces
Coelicolor: Molecular Cloning and Characterization of a New Sesquiterpene
Synthase” J. Am. Chem. Soc. 2006, 128, 6022 - 6023.

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ACKNOWLEDGEMENTS

First and for most I would like to express my appreciation to my advisor, Prof. David
E. Cane, for his teaching and guidance during my Ph.D. study. His devotion an insight to
science has always inspired me.
Thank all of other faculty and staff in Chemistry Department at Brown University who
have afforded generous help to me. Thank Prof. Amit Basu and Prof. Jason K. Sello
especially for being in my committee for the final defense, and Prof. William J. Suggs for
RPD.
Thank Tun-Li Shen and Russell E. Hopson for your assistance with all my GC-MS
and NMR problems. They are always there whenever I need help.
I would like to thank all of my colleagues in Prof. Cane’s research group for all of the
encouragement they gave me during the years. Also thank all of my friends at Brown
University for their understanding and care in my life. Especially I would like to thank
Xinyuan Liu for her generous help in my life during the past years.
I am deeply grateful to my parents for their everlasting love and support. Their
kindness has always been a inspiration to me. Last but not least, thank Jin Xie. He is
always there for me, throughout happiness or depression. He taught me the importance of
sharing and forgiving. And only with your involvement, that this moment becomes
meaningful.

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To Mon and Dad

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Abstract of “Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone”, by Xin Lin,
Ph.D., Brown University, May 2009.

Streptomyces are Gram-positive, filamentous, soil-dwelling saprophytic bacteria that
are the source of a vast array of bioactive secondary metabolites. The albaflavenone
biosynthetic gene cluster in S. coelicolor A3(2) contains two ORFs, sco5222 and sco5223.
The 1086-bp sco5222 gene encodes a protein of 361 amino acids (aa), which has been
expressed in E. coli and characterized to catalyze the cyclization of farnesyl diphosphate
(FPP) to epi-isozizaene. The sco5223 gene shares a four-nucleotide ATGA transcriptional
overlap with sco5222 at its 5'-end, and was demonstrated to catalyze the two-step allylic
oxidation of epi-isozizaene to albaflavenone through the intermediacy of an epimeric
mixture of albaflavenols. The incubation experiments of labeled FPP and NPP with
SCO5222 protein not only confirmed the intermediacy of (3R)-NPP in the cyclization
mechanism, but also shed valuable new light on the stereochemistry of other reaction
intermediates. In addition, the site-directed mutagenesis gave rise to the affected
distribution of aberrant products, providing strong support for the presence of proposed
intermediates. Recombinant SAV3032 in S. avermitilis has been expressed in E. coli and
refolded from inclusion bodies, and the purified protein was characterized to catalyze the
cyclization of FPP to epi-isozizaene. This result therefore implied the presence of
albaflavenone biosynthesis pathway in S. avermitilis.
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Table of Contents

Chapter I: Introduction
1.1 Sesquiterpenes and sesquiterpene synthases……………………………..........1
1.2 Cyclization mechanisms of sesquiterpene synthases…………………….........2
1.2.1. Trichodiene synthase……………………………………………….3
1.2.2. Aristolochene Synthase…………………………………………………5
1.2.3. Epi-Aristolochene Synthase……………………………………….........7
1.2.4. Pentalenene synthase……………………………………………….8
1.3 Cytochromes P450 in Streptomyces……………………………………...10
1.4 Albaflavenone…………………………………………………………..........11
Reference………………………………………………………………………...13
Chapter II Molecular Cloning and Characterization of Epi-isozizaene Synthase in
Streptomyces coelicolor A3(2)
2.1. Introduction…………………………………………………………….........16
2.2. Results and Discussion………………………………………………….17
2.3. Experimental Procedures
2.3.1. Materials and Methods………………………………………………...23
2.3.2. Preparation of farnesyl diphosphate…………………………………...24
2.3.3. Preparation of [1, 1-2H ]farnesyl diphosphate………………………...26
2
2.3.4. Preparation of (1S)-[1-2H]-farnesyl diphosphate………………..........28
2.3.5. Preparation of (1S)-[1-2H]-farnesyl diphosphate……………………..29
2.3.6. Cloning of epi-isozizaene synthase (SCO5222)………………………30
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2.3.7. Expression and purification of epi-isozizaene synthase (SCO5222) in E.
coli…………………………………………………………...33
2.3.8. Isolation, purification and identification of the sesquiterpene product
…………………………………………………………………………33
2.3.9. NMR of epi-isozizaene……………………………………………35
2.3.10. Metal cofactor requirement and kinetics test………………………...43
2.3.11. Comparison of enzymatically-generated product with synthetic
epi-isozizaene and isozizaene……………………………………45
Reference………………………………………………………………...46
Chapter III. Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone in
Streptomyces coelicolor A3(2)
3.1. Introduction………………………………………………………….48
3.2. Results and Conclusion……………………………………………...............49
3.3. Experimental Procedures
3.3.1. Materials and Methods……………………………………………….56
3.3.2. Expression and Purification of CYP170A1………………………….56
3.3.3. Expression and Purification of E. coli Flavodoxin…………………..57
3.3.4. E. coli Flavodoxin Reductase……………………………………......58
3.3.5. Incubation with epi-isozizaene………………………………………59
3.3.6. Incubation with [11, 11]-2H -epi-isozizaene…………………….......59
2
3.3.7. Synthesis of albaflavenols and albaflavenone……………………….61
3.3.8. NMR of albaflavenols and albaflavenone……………………………64
Reference……………………………………………………………….......69
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Chapter IV. Epi-isozizaene biosynthesis and the role of nerolidyl diphosphate in the
enzymatic cyclization of farnesyl diphosphate
4.1. Introduction………………………………………………………………….70
4.2. Results and discussion
4.2.1. Role of nerolidyl diphosphate (NPP)…………………………….........72
4.2.2. Cyclization stereochemistry…………………………………………...78
4.2.3. Epi-isozizaene synthase generates a mixture of sesquiterpenes………82
4.2.4. Site-directed mutagenesis of epi-isozizaene synthase…………….85
4.3. Experimental Procedures
4.3.1. Materials and Methods……………………………………………….94
4.3.2. Preparation of chiral NPPs………………………………………...95
4.3.3. Analysis of the stereochemistry of the conversion of NPP to
epi-isozizaene…………………………………………………….102
4.3.4. Calculation of the specificity preference of epi-isozizaene synthase for
(3S)-NPP versus (3R)-NPP………………………………………104
4.3.5. Incubation of (3RS)-(Z)-[1-2H]NPP with recombinant SCO5222.105
4.3.6. Incubation of (3S)-NPP with recombinant SCO5222………………106
4.3.7. Incubation of [12,12,12-2H ]FPP and [13,13,13-2H ]FPP with
3 3
epi-siozizaene synthase…………………………………………..106
4.3.8. Site-directed mutagenesis of SCO5222……………………………106
4.3.9. Expression in E. coli and purification of SCO5222 mutants…..….108
4.3.10. Isolation and identification of the sesquiterpene products from
SCO5222 mutants………………………………………………109
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