Discovery and Analysis of Novel Biochemical
Transformations
Linda J. Broadbelt
Department of Chemical and Biological Engineering
Northwestern University
Evanston, IL 60208
How Can We Create Products from
Natural Resources?
•Concern over dwindling petroleumbased resources sparks exploration
of alternative feedstocks
www.clemson.edu/edisto/ corn/corn.htm
•Biochemical processes are being
explored as alternatives to traditional
chemical processes
www.timberland.com/.../ tim_
product_detail.jsp?OID=18298
Overall reaction
biomass
polysaccharides
monosaccharides
glucose
ethanol
Reaction Networks of Novel Biochemical
Transformations
•Reactants, intermediates
and products
DG1
k1
•Thermodynamic parameters
DG9
DG6
k6
k5
DG4
k4
•Kinetic parameters
k2
DG5
•Reactions
DG3
k3
DG7 DG8
k7 k8
DG11
k11
DG10
k10
k9
DG16
k16
DG13
DG14
k14
DG15
k15
k13
DG12
k12
DG18
k18
DG17
k17
Challenges for Reaction Network
Development
Reactive intermediates have not been detected
Pathways have not been elucidated experimentally
Thermodynamic and kinetic parameters are unknown
Reaction networks are large
Construction is tedious and prone to user’s bias and
errors
Computer generation of reaction networks
Elements of Computer Generated
Reaction Networks
• Graph Theory
• Reaction Matrix
Operations
• Connectivity
Reactants
Scan
Reaction • Uniqueness
Types
Determination
Reaction • Property
Rules
Calculation
• Termination
Criteria
DG1
k1
k2
DG5
k5
DG4
k4
DG9
DG3
k3
DG6
k6
DG7 DG8
k7 k8
DG11
k11
DG10
k10
k9
DG16
k16
DG13
DG14 k13
k14
DG15
k15
DG12
k12
DG18
k18
DG17
k17
Bond-Electron Representation Allows
Implementation of Chemical Reaction
C
H
H
H
H
01111
10000
10000
10000
10000
methane
C 1111
H 1000
H 1000
H 1000
methyl radical
C
C
H
H
H
H
021001
200110
100000
010000
010000
100000
ethylene
ij entries denote the bond order between atoms i and j
ii entries designate the number of nonbonded electrons
associated with atom i
Chemical Reaction as a Matrix
Addition Operation
H • + CH4
•CH3 + H2
Reactant
Matrices
C
H
H
H
H
01111
10000
10000
10000
10000
H• 1
C
H
H
H
H
H•
Reaction Operation
H 001
0 -1 1
H 010
C• 0 1 0
C 1 0 0 + -1 1 0
H 100
1 0 -1
H• 0 0 1
Reactant
Matrix
Reordered
Reactant Matrix
011110
100000
100000
100000
100000
000001
H
C
H•
H
H
H
010
100
001
010
010
010
000
111
000
000
000
000
Product
Matrix
H
C•
H
H
H
H
001
010
100
010
010
010
000
111
000
000
000
000
Unique Patterns of Observed Enzyme Chemistry
EC i.j.k.l → unique enzyme
i → main class
j → functional group
k → cofactor / cosubstrate
Tipton, S.B. and Boyce, S. Bioinformatics. 16 (2000), 34-40
Kanehisa, M. and Goto, S. Nucleic Acid Research. 28 (2000), 27-30
4th level is specific to substrate
Formulation of Reaction Matrices
Using Enzyme Classification System
Enzyme commission (EC) code number provides
systematic names for enzymes
EC i.j.k.l
unique enzyme
i
the main class
j
the specific functional groups
k
cofactors
l
specific to the substrates
Formulation of Reaction Matrices
Using Enzyme Classification System
Enzyme commission (EC) code number provides
systematic names for enzymes
EC i.j.k.l
unique enzyme
i
the main class
j
the specific functional groups
k
cofactors
l
specific to the substrates
Generalized Enzyme Function
Examined at the i.j.k Level
•More than 5,000 specific enzyme functions
(i.j.k.l)
•Fewer than 250 generalized enzyme
functions (i.j.k)
•Novel enzyme functions should be
expected through genomic sequencing,
proteomics and protein engineering
Example of a Generalized Enzyme
Reaction
Generalized
enzyme reaction
(EC 4.2.1)
• EC 4.2.1.2 (fumarate hydratase)
OH
CO2H
HO2C
HO2C
CO2H
+
O
H
H
H-C-C-O-H
- - - -
• EC 4.2.1.3 ( aconitate hydratase)
HO
HO2C
CO2H
CO2H
HO2C
CO2H
CO2H
+
O
H
H
C=C + H2O
Matrix Representation of Generalized
Enzyme Function (i.j.k)
Generalized enzyme reaction EC 4.2.1
OH
H O 2C
CO 2 H
HO 2C
+
O
H
H
C=C + HOH
H -C -C -O -H
H
0 C
1 C
0 O
0
H 1 0 1 0
C 0 1 0 1
C 0 0 1 4
O
Reactant
CO 2H
+
C
-1
0
1
0
C
0
1
0
-1
O
1
0
-1
0
H0
H -1
=
C 0
C 1
O
Reaction operator
O
1
0
0
4
H
0 C
0 C
0
H 0 0 2
C 0 2 0
C 1 0 0
O
Products
Discovery of Novel Biosynthetic Routes
I.J.K
A+B
C
I.J.K
L.M.N
Q.R.S
A+B
L.M.N
I.J.K
L.M.N
Q.R.S
C
D
I.J.K
L.M.N
Q.R.S
C
+A+B
Q.R.S
E
C
+A+B
D
D
E
Generation Generation
0
1
Generation
2
Generation
3
Implications for Novel Pathway
Development
Given a novel reaction (reactant/product),
can we identify enzymes (catalysts) that
could be engineered (evolved) to carry this
novel biotransformation ?
If A gives B under 2.4.1 action,
then target enzymes within the 2.4.1 class
Application of Reaction Matrix Approach
Step 1
Enumerate all enzymes in the EC system
Step 2
Choose a specific pathway to explore its
synthetic ability
Example
Aromatic amino acid biosynthesis
Exists in higher plants and microorganisms
Pathway does not exist in mammals
Aromatic Amino Acid Biosynthesis:
Phenylalanine and Tyrosine
chorismate
phenylpyruvate
glutamate
prephenate
dehydratase
chorismate
mutase
aromatic
aminotransferase
4-hydroxyphenyl
pyruvate
prephenate
dehydrogenase
prephenate
phenylalanine
glutamate
tyrosine
Aromatic Amino Acid Biosynthesis:
Phenylalanine and Tyrosine
chorismate
phenylpyruvate
glutamate
2.6.1.57
5.4.99.5
chorismate
mutase
prephenate
dehydratase
4.2.1.51
1.3.1.12
prephenate
aromatic
aminotransferase
4-hydroxyphenyl
pyruvate
prephenate
dehydrogenase
phenylalanine
2.6.1.57
glutamate
tyrosine
Reaction Misclassification (?)
Some reactions within classes are not general
General 4.2.1 reaction (4. = lyase)
Loses water (4.2.1 = hydrolyase) AND forms a
double bond. However…
4.2.1.51
prephenate
dehydratase
+ H2O + CO2
It is both a carboxy-lyase (4.1.1)
and a hydro-lyase (4.2.1)
Reaction Decomposition
4.2.1.51
prephenate
dehydratase
+ H2O + CO2
4.2.1.51 can be broken down into 3 general reactions:
4.1.1 will decarboxylate
(4.1.1 is a carboxy-lyase)
5.3.3 will rearrange the double bond
(5.3.3 transposes C=C bonds)
4.2.1 will lose H2O and form a double bond
(4.2.1 is a hydro-lyase)
Mapping Results
•Although only 2500 reactions in the KEGG and 269 reactions in the iJR904 model were
contained in the curated EC classes, 3267 (50%) of the KEGG reactions and 430 (46%)
of the iJR904 reactions were reproduced using the 86 reaction rules
•The reproduced reactions are involved in 129 different third-level enzyme classes in the
KEGG and iJR904
•100% of the reactions contained in 25 of the uncurated EC classes in the KEGG were
mapped to the 86 existing reaction rules
Tryptophan Biosynthesis Pathway
Input Molecules
phosphoenolpyruvate (PEP), erythrose-4-phosphate (E4P), glutamine, serine, ribose-5phosphate (R5P)
Cofactors
ATP, NADPH
Specific Enzyme Actions
12
The Evolution and Wealth of the Aromatic
Amino Acid Biochemistry
Number of Products
105
104
TRP
103
PHE
TYR
102
Convergence
10
1
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Generation Number
Specialty Organic Chemicals
7-carboxyindole
3-Hydroxypropanoate from Pyruvate
• 3-Hydroxypropanoate is a useful chemical with known biochemical
production routes
• Generate all of the possible compounds and reactions from
pyruvate using only the reaction rules involved in the known
biosynthetic routes to 3HP
• Generate all of the possible compounds and reactions from
pyruvate using all of the 86 current reaction rules
Number of pathways
Novel Biosynthetic Pathways Discovered:
Pyruvate to 3HP
pathways discovered using only the
reaction rules involved in the known
pathways to 3HP
pathways discovered using all
reaction rules
Distribution of lengths of pathways
106
105
104
103
102
10
1
2
3
4
5
6
7
8
Pathway length
A pathway of length two and a pathway of length three were both
discovered using the additional reaction rules
9
10
3HP-CoA
Propan-2-ol
Propane-1,3-diol
3-HP
Acryloyl-CoA
3-hydroxypropanol
Propenoate
Propene
Allyl
alcohol
Lactoyl-CoA
3-Oxopropionyl-CoA
2-hydroxy-2,4Lactate
Propane 1,2 diol
pentadienoate
Propane-1-ol
Ethylamine
Ethanol
Acetaldehyde
Malonate semialdehyde
Ethylene
Propanoyl-CoA
Alanine
Malate
Pyruvate
Fumarate
Aspartate
Propanoate
Beta-alanyl-CoA
Oxaloacetate Beta-alanine
Propanoate
Homoserine
Theonine
Acrolein
Hydroxyacetone
Lactaldehyde
3HP-CoA
3-HP
Acryloyl-CoA
Lactoyl-CoA
Lactate
Malonate semialdehyde
Pyruvate
Aspartate
Beta-alanyl-CoA
Oxaloacetate Beta-alanine
What Screening Methods Can We Use to
Identify the Most Attractive Pathways?
•Pathway length
•Fewest novel intermediates
•Thermodynamic feasibility
•Maximum achievable yield to 3HP from glucose
during anaerobic growth
•Maximum achievable intracellular activity at which
3HP can be produced
•Protein docking calculations
•Quantum chemical investigations
Shortest Novel Pathways to 3HP
CO2
glu
Oxaloacetate
4.1.1 Rev
Pathway
2-oxo
H+
nadh
2-oxo
N1
CO2
Alanine
4.1.1
Aspartate
2.6.1
glu
2-oxo
Ethylamine
4.1.1
1.1.1
Pyruvate
glu
Acetaldehyde CO
2
2.6.1
CO2
nad
4.2.1 Rev
Lactate
NH3
4.3.1
Propenoate
CoA
Pathway N2
NH3
CoA
2-oxo
2.6.1
2.3.1
H2O
Β-alanine
H2O
CoA
H2O
Lactoyl
CoA
Β-alanyl
CoA
H2O
glu
Malonate
Semialdehyde
CoA
H+
H+
nadh
nadh
H2O
2-oxo
nadh
H2O
Acryloyl
CoA
NH3
H+
nad
3-oxopropionyl
CoA
1.1.1
4.2.1
glu
nad
nad
H2O
H2O
3-HP-CoA
CoA
2.3.1 Rev
4.2.1 Rev
3-HP
KEGG Reaction not in Patented Pathways
Part of the Patented Pathway
Not found in KEGG or Patented Pathways
Attractive Novel Pathways Successfully
Identified
•Two-step pathway identified with only one novel
reaction
•Maximum achievable yield to 3HP from glucose
during anaerobic growth matches commercial pathway
•Slightly reduced maximum achievable intracellular
activity at which 3HP can be produced
•Numerous other attractive candidates
Are These Novel Reactions Feasible?
Decarboxylation reaction of ketoacids
PFOR (1.2.7.a) : pyruvate + CoA + Fd (ox)
CO2+ acetyl-CoA + Fd (red)
Generalized enzyme operators can act on all of the above keto acids to
give their corresponding products
Can the enzyme that catalyzes decarboxylation of
pyruvate perform catalysis of different substrates?
Explore Novel Reactions Using
Molecular Modeling
• Substrate binding
Docking analysis
• Ability to form initial enzyme-substrate bound
species with no distortion to the active site of the
enzyme or the cofactor
QM/MM structural studies
• Follow the reaction pattern of the native substrate
Study of reaction mechanism using QM methods
Enzyme Docking Results
Scored using GLIDE
PFOR
Substrate
1.2.7
pyruvic acid
-10.7
2-ketobutyric acid
-11.63
2-ketoisovaleric acid
-11.56
2-ketovaleric acid
-11.31
2-keto-3-methylvaleric acid
-11.27
2-keto-4-methylpentanoic acid
-11.01
phenylpyruvic acid
X
Enzyme Docking Poses
1
2
pyruvic acid
3
2-ketobutyric acid
2-ketoisovaleric acid
4
5
6
2-ketovaleric
acid
2-keto-3-methylvaleric acid
2-keto-4-methylpentanoic acid
Binding Using Quantum
Mechanics/Molecular Mechanics
MM part : 50 Å of active site and solvent molecules ~20,000 atoms
QM part : 63 atoms
Geometry : B3LYP/6-31G*
Comparison of Bound Structures of
Different Acids: QM/MM
pyruvic acid
2-ketoisovaleric acid
2-keto-3-methylvaleric acid
2-keto-4-methylpentanoic acid
QM/MM structural studies suggest that the binding of the
substrates does not cause distortions to the active site
Kinetics of Enzyme-Catalyzed
Decarboxylation: Quantum Mechanics
LThDP
TS1
TS2
HEThDP enamine
ThDP ylide + KA
Ville et al., Nature Chemical Biology, 2(6), 2006, 324
Free Energy Surface of Thiamine-Catalyzed
Decarboxylation: Pyruvic Acid
gas phase
Relative free energy ( Kcal/Mol)
15
water
dichloro ethane
TS 1
10
5
TS 2
0
-5
-10
-15
-20
-25
ThDP + pyruvic acid
-30
Reaction coordinate
LThDP
enamine + CO2
Comparison of Thiamine-Catalyzed
Decarboxylation
C-C bond formation barrier
35
Energy barrier (kcal/mol)
30
25
20
15
10
5
0
Free energy barrier (∆Gactivation298K, DCE)
C-C bond breaking barrier
Exploring Novel Pathways and Molecules
New routes to
bioavailable species
New molecules
HO
HO CO2H
HO
OH
OH
1,3,4,5-TetrahydroxyCyclohexanecarboxylic acid
O
N
H
CO2H
CO2H
3-[1-Carboxy-2-(1,4-dihydro-pyridin
-3-yl)-ethoxy]-4-hydroxy-cyclohexa-1,5-dienecarboxylic acid
Present in KEGG
(Kyoto Encyclopedia of Genes
and Genomes)
NOT present in KEGG
NOT present in CAS REGISTRY
Migration to Biocatalytic Processes
New biochemical routes
to existing chemicals
HO CO2H
HO
OH
O
1,3,5-Trihydroxy-4-oxo-cyclohexane
carboxylic acid
NOT present in KEGG
Present in CAS REGISTRY
Acknowledgments
Funding
•Department of Energy
•National Science Foundation Cyber-enabled Discovery
and Innovation
Collaborators
•Vassily Hatzimanikatis
•Chunhui Li
•Chris Henry
•Goran Krilov
•Raj Assary