Table Of ContentUnderstanding The Biosynthesis And
Utilization Of Non-Proteinogenic Amino
Acids For The Production Of Secondary
Metabolites In Bacteria
Author: Carl Victor Christianson
Persistent link: http://hdl.handle.net/2345/967
This work is posted on eScholarship@BC,
Boston College University Libraries.
Boston College Electronic Thesis or Dissertation, 2008
Copyright is held by the author, with all rights reserved, unless otherwise noted.
Boston College
The Graduate School of Arts and Sciences
Department of Chemistry
UNDERSTANDING THE BIOSYNTHESIS AND UTILIZATION OF
NON-PROTEINOGENIC AMINO ACIDS FOR THE PRODUCTION
OF SECONDARY METABOLITES IN BACTERIA
A dissertation
by
CARL VICTOR CHRISTIANSON
Submitted in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
December 2008
© Copyright by CARL VICTOR CHRISTIANSON
2008
Acknowledgements
I would like to than the members of my thesis defense committee, Jianmin Gao,
Larry McLaughlin, and James Morken for taking time to critically read this thesis and to
attend my defense.
I am grateful to Professor Steven Bruner for accepting me into his lab and for his
guidance over the past 5 years. Steve fostered an atmosphere of scientific discussion that
challenged me to constantly learn new things. I admire Steve’s steady demeanor and am
grateful to him for keeping me grounded and headed in the right direction.
I would like to thank Timothy Montavon for carefully reviewing this thesis. Tim
also contributed the synthetic projects that benefited the work presented in this thesis.
Tim has always been there for helpful scientific discussions in the four years that we have
been working together.
I have been fortunate to work with a good group of students in the Bruner lab. I
would like to thank all the current and past members of the Bruner group. I would also
like to than Deb Lynch who has helped me tremendously during my time at BC. I
believe that the whole department would fall apart without her efforts. I thank her for her
time, and for always having an open ear and full bowl of candy!
Without the help and understanding of my family, I would not have been able to
make it through these past five years. In particular I would like to thank my mother and
father for always being there and believing in me.
Finally, I would like to thank my wife, Jolene. It is through her love and
understanding that all this has been possible. I love you and to you I dedicate this thesis.
iv
Table of Contents
Chapter 1. A Review of Cofactor Independent Electrophilic Catalysis
1.1 Introduction 2
1.2 History of MIO 4
1.3 Ammonia Lyases 7
1.4 Structural Investigations of PAL 15
1.5 The Significance of (cid:1)-Amino Acids 24
1.6 MIO Dependent Aminomutases 29
1.7 Green Fluorescent Protein and Its Relation to MIO Enzymes 37
Chapter 2. The Structure of L-Tyrosine 2,3-Aminomutase From the
C-1027 Enediyne Biosynthetic Pathway
2.1 Introduction 51
2.2 Crystallization and Structure Determination 56
2.3 The Overall Structure of SgTAM 57
2.4 The Active Site Containing MIO 59
2.5 Comparison of the SgTAM Active Site with Tyrosine Ammonia Lyase 64
2.6 Restricted Active Site of SgTAM 64
2.7 Recognition of the Substrate Tyrosine in SgTAM 65
2.8 Mechanistic Implications from the Structure of SgTAM 67
2.9 Comparison of SgTAM Active Site with Green Fluorescent Protein 68
v
Chapter 3. The Mechanism of L-Tyrosine 2,3-Aminomutase and
MIO Dependent Enzymes
3.1 Introduction 82
3.2 Mechanistic Probes of SgTAM 83
3.3 Synthesis of SgTAM Substrate Analogs 86
3.4 Inhibition Studies of the Substrate Analogs of SgTAM 87
3.5 Crystallization of SgTAM in the Presence of Substrate Analogs 88
3.6 Overall Structures of SgTAM bound to Analogs 89
3.7 Structures of Product Analogs Covalently Bound to MIO 89
3.8 Epoxide Analogs Bound to SgTAM 92
3.9 Mechanistic Conclusions of MIO Dependent Enzymes 93
Chapter 4. Investigation of LnmQ
4.1 Introduction 110
4.2 Leinamycin 112
4.3 Cloning of LnmQ, a D-Ala Specific A-Domain 113
4.4 Isolation and Purification of LnmQ 115
4.5 Selenomethionine preps of LnmQ 116
4.6 Substrate and Substrate Analog Studies with LnmQ 118
Chapter 5. Conclusions and Future Directions
vi
Table of Abbreviations
A Adenine
AIP 2-aminoindan-2-phosphonate
Ala Alanine
Arg Arginine
Asn Asparagine
(cid:2)(cid:1)ME beta-mercaptoethanol
C Cytosine
Cys Cysteine
DMSO Dimethylsulfoxide
DNA (cid:2)-D-2’-Deoxyribofuranosyl Nucleic Acid
dNTPs Deoxyribonucleotide Triphosphates
DTT Dithiothreitol
G Guanine
GFP Green Fluorescent Protein
Gly Glycine
HAL Histidine Ammonia Lyase
HCA Hydroxycinnamic Acid
His Histidine
HPLC High Pressure Liquid Chromatography
MIO 4-methylideneimidazole-5-one
vii
NRP Non-Ribosomal Peptide
NRPS Non-Ribosomal Peptide Synthetase
OPA o-Phtalaldehyde
PAL Phenylalanine Ammonia Lyase
PAM Phenylalanine Aminomutase
PCR Polymerase Chain Reaction
Phe Phenylalanine
PKS Polyketide Synthase
PLP Pyridoxal Phosphate
SAM S-Adenosylmethionine
Se-Met Selenomethionine
Ser Serine
Sg Streptomyces globisporus
T Thymine
TAL Tyrosine Ammonia Lyase
TAM Tyrosine Aminomutase
TFA Trifluoroacetic Acid
THF Tetrahydrofuran
TMAO Trimethylamine-N-oxide
Tyr Tyrosine
viii
Understanding The Biosynthesis And Utilization Of Non-Proteinogenic
Amino Acids For The Production Of Secondary Metabolites In Bacteria
Carl Victor Christianson Professor Steven D. Bruner
October 24, 2008 Research Advisor
Abstract
Bacteria utilize complex enzymatic machinery to create diverse secondary
metabolites. The architectural complexities of these small molecules are enhanced by
nature’s ability to synthesize non-proteinogenic amino acids for incorporation into these
scaffolds. Many of these natural products are utilized as therapeutic agents, and it would
be advantageous to understand how the bacteria create various non-natural amino acid
building blocks. With a greater understanding of these systems, engineering could be
used to create libraries of potentially useful natural product analogs.
The tyrosine aminomutase SgTAM from the soil bacteria Streptomyces
globisporus catalyzes the formation of (cid:1)-tyrosine in the biosynthestic pathway of the
antitumor enediyne C-1027. SgTAM is homologous to the histidine ammonia lyase
family of enzymes whose activity is dependent on the methylideneimidazole-5-one
(MIO) prosthetic group. Unlike the lyase enzymes, SgTAM catalyses additional
chemical transformations resulting in an overall 2,3 amino shift in the substrate L-
ix
tyrosine to generate (S)-(cid:1)-tyrosine. The precise mechanistic role of MIO in this novel
family of aminomutases has not been established. We report the first X-ray crystal
structure of an MIO based aminomutase and confirm the structural homology of SgTAM
to ammonia lyases. Further work with mechanistic inhibitors provide structural evidence
of the mechanism by which MIO dependent enzymes operate.
We have also investigated LnmQ, an adenylation domain in the biosynthetic
pathway of leinamycin. Leinamycin is an antitumor antibiotic that was isolated from soil
samples in 1989. LnmQ is responsible for the specific recognition of D-alanine and
subsequent activation as an aminoacyl adenylate species. We have cloned the gene into a
DNA vector and expressed it in E. coli. Upon purification of the protein, crystallization
conditions have been tested. Synthesis of an inhibitor that mimics the aminoacyl
adenylate product catalyzed by LnmQ has been completed. Crystallization with this
inhibitor will provide better quality crystals and a catalytically informative co-complex.
x
Description:Larry McLaughlin, and James Morken for taking time to critically read this thesis and .. mutants where the active site serine was replaced with either an alanine or threonine, . Organisms such as Zea mays (maize) contained .. phenylpropanoid metabolism catalyzed by phenylalanine ammonia lyase.
