Fundamentals in Chemical Biology

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Fundamentals in Chemical Biology
Methods

• How to study a natural product pathway
• Chemical
• Genetic
• Biochemical

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Fundamentals in Chemical Biology
Methods
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Fundamentals in Chemical Biology
Methods
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Fundamentals in Chemical Biology
ORF-11
ClaR
CAD
ORF-10
ORF-12
PAH
CS2
ORF2
ORF-3
ORF-6
ORF-7
A
B
C
D
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Fundamentals in Chemical Biology
Clavulanate
Clavams
Streptomyces clavuligerus
Penicillin N
Cephamycin C
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Fundamentals in Chemical Biology
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Fundamentals in Chemical Biology
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Fundamentals in Chemical Biology
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Fundamentals in Chemical Biology
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Fundamentals in Chemical Biology
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Fundamentals in Chemical Biology
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Fundamentals in Chemical Biology
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Fundamentals in Chemical Biology
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Fundamentals in Chemical Biology
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Fundamentals in Chemical Biology
Methods
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Fundamentals in Chemical Biology
Methods
Analyze the chemical structure of the molecule of
interest and, based on biochemical precedent,
propose a pathway for its assembly.
serine
Polyketide
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Fundamentals in Chemical Biology
Methods
Analyze the chemical structure of the molecule of
interest and, based on biochemical precedent,
propose a pathway for its assembly.
serine
Polyketide
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Fundamentals in Chemical Biology
Methods
Establish the precursors and verify biogenic
hypothesis by feeding isotopically labeled
precursors and intermediates and observing
specific incorporation.
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Fundamentals in Chemical Biology
Methods

• Isotopes
• Radioactive 14C, 3H, 32P, 35S
• inexpensive
• easy to detect radiography, scintillation, etc.
• highly sensitive
• Non radioactive 13C, 2H, 18O, 15N
• widely available
• easy to analyze position of incorporation
• safe

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Fundamentals in Chemical Biology
Radio isotopes case study chiral acetate
Q What is the stereochemistry of addition to
acetate for malate synthase?
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Fundamentals in Chemical Biology
Radio isotopes case study chiral acetate
A Make chiral acetate
Townsend, C.A. et al. J. Chem. Soc. Chem. Comm.
921-2 (1975)
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Fundamentals in Chemical Biology
Radio isotopes case study chiral acetate
Results
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Fundamentals in Chemical Biology
Stable isotope labeling
Non radioactive 13C, 2H, 18O, 15N
Natural isotopic abundances 1H 99.985 2H
.0015 12C 98.90 13C 1.10 16O 98.76 18O
0.2 14N 98.63 15N 0.37
Need to know incorporation position of
incorporation
NMR Mass Spectrometry
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Fundamentals in Chemical Biology
Mass spectrometry case study morphine
biosynthesis
Surprising result Human neurons seem to be able
to make their own morphine!
Proof Tandem MS spectrometry
steps
Proc Natl Acad Sci U S A. 2005 Jun
14102(24)8495-500.
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Fundamentals in Chemical Biology
Mass spectrometry case study morphine
biosynthesis
Tandem Mass Spectrometry
Examine incorporation into molecule and
fragments
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Fundamentals in Chemical Biology
Mass spectrometry case study morphine
biosynthesis
(Note these are product ion spectra, actually 2
incorporation)
Proc Natl Acad Sci U S A. 2004 Sep
28101(39)14091-6.
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Fundamentals in Chemical Biology
Mass spectrometry case study morphine
biosynthesis
Positional discrimination is possible by
MS/MS Scan for enrichment in known MS/MS
fragments of morphine shows that tyramine goes
into the cyclohexene ring of morphine only.
Proc Natl Acad Sci U S A. 2004 Sep
28101(39)14091-6.
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Fundamentals in Chemical Biology
NMR for isotopic incorporation
Natural isotopic abundances 1H 99.985 2H
.0015 12C 98.90 13C 1.10 16O 98.76 17O
0.04 18O 0.2 14N 98.63 15N 0.37
Natural isotopic abundances 1H 99.985 2H
.0015 12C 98.90 13C 1.10 16O 98.76 17O
0.04 18O 0.2 14N 98.63 15N 0.37
Natural isotopic abundances 1H 99.985 2H
.0015 12C 98.90 13C 1.10 16O 98.76 17O
0.04 18O 0.2 14N 98.63 15N 0.37
Natural isotopic abundances 1H 99.985 2H
.0015 12C 98.90 13C 1.10 16O 98.76 17O
0.04 18O 0.2 14N 98.63 15N 0.37
NMR Active
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Fundamentals in Chemical Biology
Case Study Aflatoxin
singlet
doublet
doublet
singlet
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Fundamentals in Chemical Biology
Case Study Methyleneomycin
(antibiotic)
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Fundamentals in Chemical Biology
Case Study Methyleneomycin
Hypothesis
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Fundamentals in Chemical Biology
Methods
Step 1 Find a gene!

• Biochemistry
• Genetics
• Random mutagenenesis
• Knowledge based approaches
• Genomics

Step 2 Find gene cluster
Step 3 Sequence
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Fundamentals in Chemical Biology
Biochemical Methods
Identify time course/location of production
Make cell-free extract
Assay for product formation
Purify enzyme
Identify gene
Once a biogenetic scheme is established by
labeling studies or hypothesis, proposed
intermediates/precursors can be used to discover
the enzymes involved.
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Fundamentals in Chemical Biology
Biochemical Methods
Identify time course/location of production
Make cell-free extract
Assay for product formation
Purify enzyme
Identify gene
Natural product genes/enzymes are
spatiotemporally regulated
Eukaryotes/Plants natural products are expressed
in plant components (e.g. leaf shoots, roots,
bark). The cells producing the natural products
need to be identified.
Bacteria Growth media and growth phase of
production need to be identified.
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Fundamentals in Chemical Biology
Biochemical Methods
Identify time course/location of production
Make cell-free extract
Assay for product formation
Purify enzyme
Identify gene
Cell contents (biosynthetic enzymes) must be
released without denaturing them
Bacteria/Fungi Ultrasonication, French Pressure
Cell
Eukaryotes/plants Grind tissue in buffer (in
sand/dry ice),
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Fundamentals in Chemical Biology
Biochemical Methods
Identify time course/location of production
Make cell-free extract
Assay for product formation
Purify enzyme
Identify gene

• Verify conversion of starting material/intermediat
  e to product
• Mass Spectrometry
• NMR
• Radiography Radiolabeled TLC followed by
  exposure to film

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Fundamentals in Chemical Biology
Biochemical Methods
Identify time course/location of production
Make cell-free extract
Assay for product formation
Purify enzyme
Identify gene

• Develop robust inexpensive assay for enzyme of
  interest
• Colorimetric (UV/Vis)
• Bioactivity
• Radiography

• Use standard protein purification methodologies
  to purify enzyme
• Ion exchange
• Hydrophobicity
• Size

End-sequence peptide by degradation or MS
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Fundamentals in Chemical Biology
Biochemical Methods
Identify time course/location of production
Make cell-free extract
Assay for product formation
Purify enzyme
Identify gene
Design degenerate PCR primers to amplify
corresponding gene
Make a degenerate radiolabeled probe to identify
gene from a genomic library
In bacteria, genes are clustered. If you find
one gene, you find them all.
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Fundamentals in Chemical Biology
Genetic approaches for NP gene discovery

• Random mutagenenesis
• Create a large random library of point mutants
  (single base mutations) by UV or carcinogen or
  transposon mutagenesis
• Screen for change in chemophenotype (loss of
  natural product production or self-resistance) to
  identify a mutant incapable of synthesizing a
  natural product of interest
• Genetically complement mutation in non-producer
  by transforming with a random genomic library and
  sequence positive clone. Or, if using
  transposon, sequence out from known transposon
  sequence

Example (Tabtoxin) J Bacteriol. 1991
Jul173(13)4124-32.
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Fundamentals in Chemical Biology
Genetic approaches for NP gene discovery

• Knowledge based cloning
• Use isolated enzyme (above) to design PCR probe
  or radio-oiligo to isolate corresponding gene.
• Use biosynthetic precedent to design PCR primers
  to amplify genes predicted to be in the pathway.
  Great precedented systems, impossible for
  entirely novel systems.

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Fundamentals in Chemical Biology
Genetic approaches for NP gene discovery
Genome scanning
6 8 MBp
Small sequence library
hot GSTs
Large Library
bookmark
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Fundamentals in Chemical Biology
Genetic approaches for NP gene discovery
1. Genomic DNA

• Genome sequencing
• Sequence whole genome
• Search for predicted genes/activities
• Annotate gene cluster

2.
40,000 bp fragments
10,000 bp fragments
40 kb
10 kb
Example Proc Natl Acad Sci U S A. 2005 May
17102(20)7315-20.
3. Sequence (800 bp runs) and assemble
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Fundamentals in Chemical Biology
Methods

• Gene/enzymes role in a biosynthetic pathway must
  be proven experimentally
• Heterologous expression of individual genes
  prove expressed enzymes performs the predicted
  activity specifically.
• 2) Heterologous expression of pathway Express
  whole pathway in clean host via cosmid, fosmid
  or BAC.
• 3) Targeted mutagenesis. Delete specific gene and
  observe accumulation of intermediates and/or loss
  of natural product. Must complement mutation
  genetically or chemically to rule out polar
  effects

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Fundamentals in Chemical Biology
Methods

• Characterize individual enzymes
• Kinetics
• Biochemical Mechanism
• Substrate specificity
• Synthetic utility?