“creating the right chemistry” in pathways master seminar fall 2006 m.a. ott (cmbi)

“Creating the right chemistry” in Pathways

Master Seminar Fall 2006

M.A. Ott (CMBI)

Main concept

”Creating the right chemistry” in PathwaysM.A. Ott

• Enzymes don’t do new chemistry;

they do ‘old’ chemistry faster

Enzymes don’t perform magic;

they perform known (organic) chemistry

Contents


• Enzymes and Metabolites

• Metabolic Pathways and Reactions

• Representing Chemistry

• BioMeta Database

Ongoing progess in “omics”


• Genomics …going strong

• Transcriptomics …good progress

• Proteomics …some progress

• Metabolomics …less progress

• Systems biology …not in ten years

Metabolite and pathway databases


http://www.genome.ad.jp/kegg/

http://www.brenda.uni-koeln.de/

http://www.ebi.ac.uk/chebi/

http://www.expasy.org/tools/pathways/

http://www.empproject.com/

http://metacyc.org/

http://ecocyc.org/

http://www.ncgr.org/pathdb/

http://medicago.vbi.vt.edu/B-Net/

http://www.mol-net.de/biopath/

http://www.ergo-light.com/ERGO/

http://www.reactome.org/

http://www.chem.qmul.ac.uk/iubmb/enzyme/

Kyoto Encyclopedia of Genes and Genomes

BRENDA enzyme database

Chemical Entities of Biological Interest

ExPASy biochemical pathway

EMP enzymology and metabolism data

MetaCyc metabolic pathways

EcoCyc E. coli genome and metabolism

PathDB biological pathways and networks

B-NET - Biochemical Network

BioPath C@ROL database

ERGO comprehensive genome analysis

Reactome biological processes

IUBMB enzymes

Metabolism and friends


• Metabolism: The reactions in organisms

• Anabolism: Synthesis of larger biomolecules

from smaller ones

» Usually requires energy input

• Catabolism: Break down of larger molecules

into smaller ones

» Usually releases energy

Enzymes


• Virtually all metabolic reactions require enzymatic

catalysis

• Rate enhancement factor can be up to 1015

• Enzymes are highly reaction-specific, catalyzing

only one conversion

• Enzymes are substrate-specific, limiting their action

to only one compound or to related compounds

The metabolic network


Some metabolites


• “Housekeeping” metabolites:

Water, NADH, ATP, Phosphate, …

• End products of pathways:

Vitamin B12, Urea, Penicillin, Ecdysone, …

• Intermediates in pathways:

Glucose-6-P, Lanosterol, Precorrins, Shikimate, …

O

O

O

O

O

O

O

O

O

O

O

H

H

H

OP

O

O O

Housekeeping metabolites: ATP / ADP


Full structures:

Shorthand:

N

N

ON

OHN

NH2

O

OH

PO

PO

POH

O O OOH OH OH

N

N

ON

OHN

NH2

O

OH

PO

POH

O OOH OH

ROH

ROH

RO

POH

O OH

RO

POH

O OH

+ +

2.7.1.10

ATP ADP

2.7.1.10

Housekeeping metabolites: NADH / NAD+


Full structures:

Shorthand:

R1

O

R2

R1

OH

R2H

PO O

O OH

PO

OH O

O

OH

O

OH

N

OHOH

N+

N

N

N

NH2

ONH2

PO O

O OH

PO

OH O

O

OH

O

OH

N

OHOH

N

N

N

N

NH2

ONH2

H+ R

1

OH

R2HR

1

O

R2

+ ++

1.1.1.63

NADH, H+ NAD+

1.1.1.63

Housekeeping metabolites and chemical distance


H

O

CH3H

OH

CH3

H

C D

NADH, H+ NAD+

CH3

O

CH3CH3

OH

CH3

H

A B

NADH, H+ NAD+

What is the chemical distance between B and D?

Typical housekeeping metabolites


WaterAmmoniaPhosphateATP, ADPNADH, NAD+

NADPH, NADP+

FAD, FADH2FMN, FMNH2SAM, SAHCoenzyme AGlutamate/2-Oxoglutarate

PhosphorylationReduction/oxidationReduction/oxidation

Reduction/oxidationReduction/oxidationMethyl group transferAcyl group transferAmino/ketone group exchange

Cholesterol biosynthesis outline

Pathways and MoleculesM.A. Ott

H

H

H

H

OH

C2

Acetate

C5

Isopentenyl-PP

C30

Lanosterol

C27

Cholesterol

C30

Squalene

C6

Mevalonate

C30

Lanosterol

C5

Isopentenyl-PPx 3

x 6

- CO2

- 2 CO2

- HCO2H

Cholesterol biosynthesis from squalene


H

H

H

H

OH

HH

H

OH

H

H

H

H

OH

CH3

HHO

H

O

H

HOH

H

H

HO

H

H

H

H

H

H

OH

Squalene

Cholesterol

Lanosterol

Zymosterol

Metabolic disorders


• Inability to synthesize compounds:

Absence of important compounds

• Inability to degrade compounds:

Accumulation of undesired waste products

E.g., in phenylketonuria

Phenylketonuria (error in phenylalanine catabolism)


N

O

O

N

O

O

O

O

O

O

O

O

O

O2 H2O

BH4 BH2

Phenylalanine Tyrosine

Phenylpyruvate Phenyllactate

NADPH, H+ NADP+

Glutamate

2-Oxoglutarate

The BioMeta Database


BioMeta database structure


BioMeta database structure


R# M# Role Stoich1 1 Substrate 11 2 Substrate 11 3 Product 11 4 Product 12 2 Substrate 12 3 Substrate 12 5 Product 12 6 Product 13 6 Substrate 23 7 Product 1

# CASREG1 1065-31-72 387-49-33 76-55-14 182-20-95 450-16-56 2754-97-27 233-04-8

# Revers1 Yes2 No3 No

Molecules

Reaction 1: Mol_1 + Mol_2 Mol_3 + Mol_4

Reaction 2: Mol_2 + Mol_3 Mol_5 + Mol_6

Reaction 3: 2 Mol_6 Mol_7R-M Links

Reactions

Sample database query


OH

O

OH

O O

O

O

O

O2 H2O2

EC 1.2.3.4+

ec_nr | enzyme_name | rxn_id |rev | role |stoich| name

--------+-----------------+----------+----+------+------+------------

1.2.3.4 | oxalate oxidase | MR000247 | ir | s | 1 | Oxalic acid

,, | ,, | ,, | ,, | s | 1 | O2

,, | ,, | ,, | ,, | p | 1 | H2O2

,, | ,, | ,, | ,, | p | 2 | CO2

Molecular structure identification


O OH

OH

OH

OH

OH

O OH

OH

OH

OH

OH

NH2

NH

NH2+

R

N

N

N

OHOH

OH

NH

NH

NH

O

O

O

H2N+

NH

NH2

R

NH2

NH+

NH2

R

• Mesomerism (resonance forms)

• Tautomerism (variable positions of hydrogens)

• Other “chemical” flexibility

• Protonation state

Molecular structures and reactions


NN

NNH

O

O

OH

OHOH

OH

POH OH

OHOP OOH

OH

O O

OH OH

OH

P OOH

OH

O

OH

OH

OH

NH

NH

NH2 NH

O

O

+ +

Reactions: balancing atoms & charges

adding direction/reversibility

Molecules: adding stereochemical configurations

“canonicalizing” tautomeric form

No stereochemistry

No stereochemistry

Phosphorus imbalance

Direction

Carbon imbalance

And now what’s really going on…


P

O

OHOH

OHOH

O

O

POH

O OH

OH

O

O

POH

O OH

OP

OH

O OH

OOH

OHOH

OH

O

H2O

NN

NNH

O

O

OH

OHOH

OH

NNH

NNH

O

O

OH

OHOH

OH

+ +

+

directionirrevers.

+

2

balance

new

stereostereo

Accuracy required!


Without accurate chemical structures and balanced

reaction descriptions, a biochemical pathways database

is unsuitable for “chemical computation”:

• Graph-theoretical considerations for creating a network

• Idem for describing fluxes

• Docking applications need correct stereochemistry

Validation results (structures)


Type of Error # in KEGG # in BioMeta # Corrected

Structure missing 1239 1106 133

Valence violation(s) 76 0 76

Incorrect constitution unknown unknown 107

Total (constitution) 1315 1106 316

Undefined stereo double bond(s) 35 32 3

Invalid sp3 stereocenter(s) 70 47 23

Ambiguous sp3 stereocenter(s) 46 0 46

Undefined sp3 stereocenter(s) 1398 865 533

Unspecified enantiomer 2326 1840 486

Undefined sp3 stereochemistry 554 366 188

Incorrect stereochemistry unknown unknown 69

Total (stereochemistry) 3990 2907 1152

Total corrected 1468

Validation results (reaction balance)

Count Perc.

Total 6089 100.00

Unknown 766 12.58

Balanced 3711 60.95

Unbalanced 1612 26.47

Corrected * 893 14.66


Difference Count

[H+] * 801

2[H+] * 35

H2O * 57

O 98

H2 17

H2 – 2[H+] 41

2H2 17

H2 – [H+] 18

CH2 30

O2 – H2O 17

Some unresolved issues


O

HO OH

OHO

PHO

OHO

D-Ribose 5-phosphate ?

O

HO OH

OHO

PHO

OHO O

HO OH

OHO

PHO

OHO

O

OH OH

PHO

OHO OH

O

O

HO OH

HN

OP

HO

OHO

N NH

H2N

O

NH2

O

HO OH

HN

OP

HO

OHO

N N

H2N

OH

NH2

2,5-Diamino-6-hydroxy-4-(5'-phosphoribosylamino)-pyrimidine ?

BioMeta database


• Based on freely available data (KEGG Ligand database)

• Augmentation, completion and correction of

small-molecule information:

- Adding missing stereochemistry to structures

- Balancing atoms and charges in reactions

- Establishing the reversibility and direction of reactions

• Search for alternative pathways between metabolites

• Search for “missing” enzymes

BioMeta: things being done


• Enhancing compound/reaction structures

• Establishing directionality/reversibility of reactions

• Distinguishing cofactors/“current”/“external”/ “common” metabolites from ‘real’ metabolites

• Adding more compound data, e.g. pKa (curve)

• Increasing functionality of WWW interface

BioMeta: things to do


• Atom-to-atom correspondence (reaction mapping)

• Tautomer handling

• Pathway searches

• Putative/hypothetical reactions for predicting

metabolism

Acknowledgements


Felix van Diggelen

Gert Vriend

CMBI

“creating the right chemistry” in pathways master seminar fall 2006 m.a. ott (cmbi)

Documents

right chemistry

ott http

ott slide

metabolites metabolic

new chemistry

ott cmbi slide

ott metabolism

ott housekeeping metabolites