Discovery and Analysis of Novel Biochemical Transformations

Linda J. Broadbelt

Department of Chemical and Biological EngineeringNorthwestern University

Evanston, IL 60208

How Can We Create Products from Natural Resources?

•Biochemical processes are being explored as alternatives to traditionalchemical processes

Overall reaction

•Concern over dwindling petroleum-based resources sparks explorationof alternative feedstocks

www.clemson.edu/edisto/ corn/corn.htm

www.timberland.com/.../ tim_product_detail.jsp?OID=18298

biomass polysaccharides monosaccharides glucose ethanol

Reaction Networks of Novel Biochemical Transformations

•Reactants, intermediatesand products

•Reactions

•Thermodynamic parameters

DG1 DG2DG3

DG4

DG5DG7

DG6DG8

DG9

DG10

DG11

DG13DG12

DG14

DG15

DG16 DG17

DG18•Kinetic parameters

k1k2

k4

k6 k7

k9

k3

k8

k5

k10

k12k13

k15

k16

k14

k17

k11

k18

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 Scan• Uniqueness Determination• Property Calculation• Termination Criteria

ReactantsReactionTypesReactionRules

k1 k2

k4

k6 k7

k9

k3

k8k5

k10

k12k13

k15k16

k14

k17

k11

k18

DG1 DG2DG3

DG4

DG5DG7

DG6DG8

DG9

DG10

DG11

DG13DG12

DG14

DG15

DG16 DG17

DG18

Bond-Electron Representation Allows Implementation of Chemical Reaction

ij entries denote the bond order between atoms i and j ii entries designate the number of nonbonded electrons associated with atom i

methane methyl radical ethylene

C 0 1 1 1 1H 1 0 0 0 0H 1 0 0 0 0H 1 0 0 0 0H 1 0 0 0 0

C 0 2 1 0 0 1 C 2 0 0 1 1 0 H 1 0 0 0 0 0 H 0 1 0 0 0 0 H 0 1 0 0 0 0H 1 0 0 0 0 0

C 1 1 1 1H 1 0 0 0H 1 0 0 0

C 1 1 1 1H 1 0 0 0H 1 0 0 0H 1 0 0 0

Reaction OperationH 0 1 0C 1 0 0H• 0 0 1

H 0 0 1C• 0 1 0H 1 0 0

+ 0 -1 1-1 1 0 1 0 -1

ReactantMatrices Reactant

MatrixReordered

Reactant MatrixProductMatrix

C 0 1 1 1 1H 1 0 0 0 0H 1 0 0 0 0H 1 0 0 0 0H 1 0 0 0 0

H• 1

C 0 1 1 1 1 0H 1 0 0 0 0 0H 1 0 0 0 0 0H 1 0 0 0 0 0H 1 0 0 0 0 0H• 0 0 0 0 0 1

H 0 1 0 0 0 0C 1 0 0 1 1 1H• 0 0 1 0 0 0H 0 1 0 0 0 0H 0 1 0 0 0 0H 0 1 0 0 0 0

H 0 0 1 0 0 0C• 0 1 0 1 1 1H 1 0 0 0 0 0H 0 1 0 0 0 0H 0 1 0 0 0 0H 0 1 0 0 0 0

H • + CH4 •CH3 + H2

Chemical Reaction as a Matrix Addition Operation

EC i.j.k.l → unique enzyme

Tipton, S.B. and Boyce, S. Bioinformatics. 16 (2000), 34-40Kanehisa, M. and Goto, S. Nucleic Acid Research. 28 (2000), 27-30

i → main class j → functional groupk → cofactor / cosubstrate

Unique Patterns of Observed Enzyme Chemistry

4th level is specific to substrate

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

Formulation of Reaction Matrices Using Enzyme Classification System

•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

Generalized Enzyme Function Examined at the i.j.k Level

Example of a Generalized Enzyme Reaction

• EC 4.2.1.2 (fumarate hydratase)

• EC 4.2.1.3 ( aconitate hydratase)H-C-C-O-H

C=C + H2O

Generalized enzyme reaction

(EC 4.2.1)

H-C-C-O-H

C=C + H2O

- - - -

+HO2CCO2H

OH

HO2CCO2H

HO

H

+HO2CCO2H

CO2HH

OH

HO2CCO2H

HO

CO2H

Matrix Representation of Generalized Enzyme Function (i.j.k)

ProductsReaction operator

+

Reactant

++HO2CCO 2H

O H

HO2CCO 2H

O H

HO2CCO2H

HO2CCO2H

HO2CCO2H

H

O

HH

O

HH

O

H

4100O

1010C

0101C

0010H

OCCH

0-101O

-1010C

010-1C

10-1H

OCC

4001O

0020C

0200C

1000H

OCCH

Generalized enzyme reaction EC 4.2.1

C=C +H -C-C-O-H

H

HOH

=0

A + B + A + B

C C

D

C

I.J.KL.M.NQ.R.S

D

+ A + B

E

Generation1

Generation2

Generation3

A + B C D EI.J.K L.M.N Q.R.S

I.J.KL.M.NQ.R.S

I.J.KL.M.NQ.R.S

Generation0

Discovery of Novel Biosynthetic Routes

Implications for Novel Pathway Development

Given a novel reaction (reactant/product),can we identify enzymes (catalysts) thatcould be engineered (evolved) to carry thisnovel biotransformation ?

If A gives B under 2.4.1 action,

then target enzymes within the 2.4.1 class

Step 1Enumerate all enzymes in the EC system

Step 2Choose a specific pathway to explore its synthetic ability

ExampleAromatic amino acid biosynthesis

Application of Reaction Matrix Approach

Exists in higher plants and microorganisms Pathway does not exist in mammals

chorismate

prephenate

phenylalanine

tyrosine

4-hydroxyphenylpyruvate

phenylpyruvate

Aromatic Amino Acid Biosynthesis: Phenylalanine and Tyrosine

aromaticaminotransferase

prephenatedehydratase

chorismatemutase

prephenatedehydrogenase

glutamate

glutamate

chorismate

prephenate

phenylalanine

tyrosine

4-hydroxyphenylpyruvate

phenylpyruvate

aromaticaminotransferase

prephenatedehydratase

chorismatemutase

prephenatedehydrogenase

glutamate

glutamate

5.4.99.5

2.6.1.57

1.3.1.12 2.6.1.57

4.2.1.51

Aromatic Amino Acid Biosynthesis: Phenylalanine and Tyrosine

Reaction Misclassification (?)

Some reactions within classes are not generalGeneral 4.2.1 reaction (4. = lyase)Loses water (4.2.1 = hydrolyase) AND forms a double bond. However…

4.2.1.51 + H2O + CO2

It is both a carboxy-lyase (4.1.1)and a hydro-lyase (4.2.1)

prephenatedehydratase

4.2.1.51 + 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)

Reaction Decomposition

prephenatedehydratase

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 Moleculesphosphoenolpyruvate (PEP), erythrose-4-phosphate (E4P), glutamine, serine, ribose-5-

phosphate (R5P)

CofactorsATP, NADPH

Specific Enzyme Actions12

The Evolution and Wealth of the Aromatic Amino Acid Biochemistry

Convergence

1

10

102

103

104

105

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Num

ber o

f Pro

duct

s

TYR

TRP

PHE

Generation Number

7-carboxyindole

Specialty Organic Chemicals

• 3-Hydroxypropanoate is a useful chemical with known biochemical production routes

3-Hydroxypropanoate from Pyruvate

• 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

2 3 4 5 6 7 8 9 10

Novel Biosynthetic Pathways Discovered: Pyruvate to 3HP

Distribution of lengths of pathways

Pathway lengthN

umbe

r of p

athw

ays

1

102

103

104

10

105

106

A pathway of length two and a pathway of length three were both discovered using the additional reaction rules

pathways discovered using all reaction rules

pathways discovered using only the reaction rules involved in the known pathways to 3HP

Pyruvate

Oxaloacetate

Alanine

Lactate

Propenoate

Ethylamine

Hydroxyacetone

Homoserine

2-hydroxy-2,4-pentadienoate

Propan-2-ol

Propene

Propane-1-ol

Propanoate

Propanoate

TheonineLactaldehyde

Acrolein

Beta-alanine

3-hydroxypropanol

3-HP

Propane-1,3-diol

AspartateFumarate

Malate

Malonate semialdehyde

Ethanol

Allyl alcohol

Propane 1,2 diol

Ethylene

Acetaldehyde Propanoyl-

CoA

3HP-CoA

Lactoyl-CoAAcryloyl-CoA

Beta-alanyl-CoA

3-Oxopropionyl-CoA

Pyruvate

Oxaloacetate

Lactate

Beta-alanine

3-HP

Aspartate

Malonate semialdehyde

3HP-CoA

Lactoyl-CoAAcryloyl-CoA

Beta-alanyl-CoA

•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

What Screening Methods Can We Use to Identify the Most Attractive Pathways?

Shortest Novel Pathways to 3HP

Part of the Patented Pathway

KEGG Reaction not in Patented Pathways Not found in KEGG or Patented

Pathways

CO2

glu 2-oxoCO2

2-oxo

glu

nad

nadhH+

nad

nadhH+

H2O

CoA

H2O

1.1.

12.

3.1

4.2.

1

4.1.1 Rev 2.6.14.1.1

2.6.11.1.1

4.2.1 Rev 2.3.1 Rev

4.1.1 2.6.14.3.1

2.6.1

4.2.1 Rev 4.3.1

2.6.14.3.1

2.3.1

2.3.1

1.1.1

4.2.1 Rev

4.1.1 Rev

H2O

NH3

H2O CoA

H2O

CoA

glu

2-oxo

CoA

H2O

glu2-oxoCO2

CO2

NH3

H2O NH3

glu

2-oxo

H2O

nad

nadhH+

Pyruvate Oxaloacetate Aspartate

Lactate Β-alanine

Β-alanylCoA

Lactoyl CoA

Acryloyl CoA

3-HP-CoA 3-HP

MalonateSemialdehyde

Propenoate

Alanine Ethylamine Acetaldehyde

3-oxopropionylCoA

Pathway N1

Pathway N2

1.1.1 & 2.3.1

nadnadh H+

CoA

•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

Attractive Novel Pathways Successfully Identified

Can the enzyme that catalyzes decarboxylation of pyruvate perform catalysis of different substrates?

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

Are These Novel Reactions Feasible?

• 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

Explore Novel Reactions Using Molecular Modeling

PFORSubstrate 1.2.7

pyruvic acid -10.72-ketobutyric acid -11.63

2-ketoisovaleric acid -11.562-ketovaleric acid -11.31

2-keto-3-methylvaleric acid -11.272-keto-4-methylpentanoic acid -11.01

phenylpyruvic acid X

Scored using GLIDE

Enzyme Docking Results

1 2

3

4 56

pyruvic acid

2-ketovaleric acid

2-keto-4-methylpentanoic acid

2-ketoisovaleric acid

2-ketobutyric acid

2-keto-3-methylvaleric acid

Enzyme Docking Poses

MM part : 50 Å of active site and solvent molecules ~20,000 atomsQM part : 63 atoms Geometry : B3LYP/6-31G*

Binding Using Quantum Mechanics/Molecular Mechanics

2-keto-4-methylpentanoic acid

pyruvic acid

2-keto-3-methylvaleric acid

2-ketoisovaleric acid

QM/MM structural studies suggest that the binding of the substrates does not cause distortions to the active site

Comparison of Bound Structures of Different Acids: QM/MM

N

S

H3C

N

N

NH2

CH3

N

S

H3C

N

N

NH2

CH3

CC

O

O

OH

C

N

S

H3C

N

N

NH2

CH3

CC

O

O

OH

E

N

S

H3C

N

N

NH2

CH3

C

C

O

O

OH

G

CC bond formation TSB

Proton Transfer TSD

C-C bond cleavage TS

FTS1 TS2

O

OH

A

HEThDP enamine

LThDP

ThDP ylide + KAVille et al., Nature Chemical Biology, 2(6), 2006, 324

Kinetics of Enzyme-Catalyzed Decarboxylation: Quantum Mechanics

-30

-25

-20

-15

-10

-5

0

5

10

15

Reaction coordinate

Rel

ativ

e fr

ee e

nerg

y ( K

cal/M

ol)

gas phase water dichloro ethane

TS 1

TS 2

ThDP + pyruvic acid

LThDP enamine + CO2

Free Energy Surface of Thiamine-Catalyzed Decarboxylation: Pyruvic Acid

Free energy barrier (∆Gactivation298K, DCE)

0

5

10

15

20

25

30

35

Ener

gy b

arrie

r (kc

al/m

ol)

C-C bond formation barrier C-C bond breaking barrier

O

OH

OO

OH

O

O

OH

O O

OH

OO

OH

O

O

O

OH

O

OH

O

Comparison of Thiamine-Catalyzed Decarboxylation

NOT present in KEGG NOT present in CAS REGISTRY

1,3,4,5-Tetrahydroxy-Cyclohexanecarboxylic acid

HO CO2H

HOOH

OH

3-[1-Carboxy-2-(1,4-dihydro-pyridin-3-yl)-ethoxy]-4-hydroxy-cyclohexa--1,5-dienecarboxylic acid

NH

O

CO2H

HO

CO2H

Present in KEGG(Kyoto Encyclopedia of Genes

and Genomes)

Exploring Novel Pathways and Molecules

New routes tobioavailable species New molecules

Migration to Biocatalytic Processes

HO CO2H

HOO

OH

NOT present in KEGGPresent in CAS REGISTRY

1,3,5-Trihydroxy-4-oxo-cyclohexanecarboxylic acid

New biochemical routesto existing chemicals

Acknowledgments

•Department of Energy•National Science Foundation Cyber-enabled Discoveryand Innovation

•Vassily Hatzimanikatis•Chunhui Li•Chris Henry•Goran Krilov•Raj Assary

Funding

Collaborators