“creating the right chemistry” in pathways master seminar fall 2006 m.a. ott (cmbi)
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“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
”Creating the right chemistry” in PathwaysM.A. Ott
• Enzymes and Metabolites
• Metabolic Pathways and Reactions
• Representing Chemistry
• BioMeta Database
Ongoing progess in “omics”
”Creating the right chemistry” in PathwaysM.A. Ott
• Genomics …going strong
• Transcriptomics …good progress
• Proteomics …some progress
• Metabolomics …less progress
• Systems biology …not in ten years
Metabolite and pathway databases
”Creating the right chemistry” in PathwaysM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
• 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
”Creating the right chemistry” in PathwaysM.A. Ott
• 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
”Creating the right chemistry” in PathwaysM.A. Ott
Some metabolites
”Creating the right chemistry” in PathwaysM.A. Ott
• “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
”Creating the right chemistry” in PathwaysM.A. Ott
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+
”Creating the right chemistry” in PathwaysM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
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
Pathways and MoleculesM.A. Ott
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
Pathways and MoleculesM.A. Ott
• 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)
Pathways and MoleculesM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
The BioMeta Database
”Creating the right chemistry” in PathwaysM.A. Ott
BioMeta database structure
”Creating the right chemistry” in PathwaysM.A. Ott
BioMeta database structure
”Creating the right chemistry” in PathwaysM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
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…
”Creating the right chemistry” in PathwaysM.A. Ott
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!
”Creating the right chemistry” in PathwaysM.A. Ott
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)
”Creating the right chemistry” in PathwaysM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
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
”Creating the right chemistry” in PathwaysM.A. Ott
• 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
”Creating the right chemistry” in PathwaysM.A. Ott
• 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
”Creating the right chemistry” in PathwaysM.A. Ott
• Atom-to-atom correspondence (reaction mapping)
• Tautomer handling
• Pathway searches
• Putative/hypothetical reactions for predicting
metabolism
Acknowledgements
”Creating the right chemistry” in PathwaysM.A. Ott
Felix van Diggelen
Gert Vriend
CMBI
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