M.A. Ott (CMBI)
“Creating the right chemistry” in Pathways
Master Seminar Fall 2006
M.A. Ott
”Creating the right chemistry” in Pathways
Main concept
• Enzymes don’t do new chemistry;
they do ‘old’ chemistry faster
Enzymes don’t perform magic;
they perform known (organic) chemistry
M.A. Ott
”Creating the right chemistry” in Pathways
Contents
• Enzymes and Metabolites
• Metabolic Pathways and Reactions
• Representing Chemistry
• BioMeta Database
M.A. Ott
”Creating the right chemistry” in Pathways
Ongoing progess in “omics”
• Genomics
…going strong
• Transcriptomics
…good progress
• Proteomics
…some progress
• Metabolomics
…less progress
• Systems biology
…not in ten years
M.A. Ott
”Creating the right chemistry” in Pathways
Metabolite and pathway databases
Kyoto Encyclopedia of Genes and Genomes
http://www.genome.ad.jp/kegg/
BRENDA enzyme database
http://www.brenda.uni-koeln.de/
Chemical Entities of Biological Interest
http://www.ebi.ac.uk/chebi/
ExPASy biochemical pathway
http://www.expasy.org/tools/pathways/
EMP enzymology and metabolism data
http://www.empproject.com/
MetaCyc metabolic pathways
http://metacyc.org/
EcoCyc E. coli genome and metabolism
http://ecocyc.org/
PathDB biological pathways and networks
http://www.ncgr.org/pathdb/
B-NET - Biochemical Network
http://medicago.vbi.vt.edu/B-Net/
BioPath C@ROL database
http://www.mol-net.de/biopath/
ERGO comprehensive genome analysis
http://www.ergo-light.com/ERGO/
Reactome biological processes
http://www.reactome.org/
IUBMB enzymes
http://www.chem.qmul.ac.uk/iubmb/enzyme/
M.A. Ott
”Creating the right chemistry” in Pathways
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
M.A. Ott
”Creating the right chemistry” in Pathways
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
M.A. Ott
”Creating the right chemistry” in Pathways
The metabolic network
M.A. Ott
”Creating the right chemistry” in Pathways
Some metabolites
•
“Housekeeping” metabolites:
Water, NADH, ATP, Phosphate, …
O O
P
O
O
O
O
H
O
•
H
End products of pathways:
O
H
Vitamin B12, Urea, Penicillin, Ecdysone, … O
O
O
O
•
Intermediates in pathways:
O
Glucose-6-P, Lanosterol, Precorrins, Shikimate, … O
O
M.A. Ott
”Creating the right chemistry” in Pathways
Housekeeping metabolites: ATP / ADP
Full structures:
R
O OH
R
P
O
OH
+
OH
+
2.7.1.10
N
OH
O OH O OH O
P
P
P
HO
O
O
O
O
HO
N
OH
N
N
OH
O OH O
P
P
HO
O
O
NH2
O
N
HO
NH2
N
OH
N
Shorthand:
R
2.7.1.10
OH
ATP
ADP
O OH
R
P
O
OH
N
M.A. Ott
”Creating the right chemistry” in Pathways
Housekeeping metabolites: NADH / NAD+
Full structures:
1
2
R
1
R
+
+
R
+
H
O
O
N
O
HO O O OH
P
P
O
O
O
HO
OH
HO
NH2
NH2
N
OH
N
+
HO O O OH
P
P
O
O
O
N
O
O
2
R
OH
1.1.1.63
NH2
H
N
+
N
O
O
N
HO
HO
OH
Shorthand:
1
2
R
R
O
1.1.1.63
NADH, H+
NAD+
1
R
H
2
R
OH
NH2
N
OH
N
N
M.A. Ott
”Creating the right chemistry” in Pathways
Housekeeping metabolites and chemical distance
H3C
H3C
CH3
O
NADH, H+
B
H
CH3
O
C
CH3
OH
NAD+
A
H
H
NADH, H+
NAD+
H
CH3
OH
D
What is the chemical distance between B and D?
M.A. Ott
”Creating the right chemistry” in Pathways
Typical housekeeping metabolites
Water
Ammonia
Phosphate
ATP, ADP
NADH, NAD+
NADPH, NADP+
FAD, FADH2
FMN, FMNH2
SAM, SAH
Coenzyme A
Glutamate/2-Oxoglutarate
Phosphorylation
Reduction/oxidation
Reduction/oxidation
Reduction/oxidation
Reduction/oxidation
Methyl group transfer
Acyl group transfer
Amino/ketone group exchange
M.A. Ott
Pathways and Molecules
Cholesterol biosynthesis outline
x3
Mevalonate
C6
Isopentenyl-PP
C5
x6
Squalene
C30
Lanosterol
C30
- 2 CO2
Acetate
C2
- CO2
Lanosterol
C30
Cholesterol
C27
- HCO2H
H
H
H
HO
Isopentenyl-PP
C5
H
M.A. Ott
Pathways and Molecules
Cholesterol biosynthesis from squalene
H
CH 3
Squalene
HO
O
H
H
H
H
H
HO
Lanosterol
H
HO
H
H
Zymosterol
H
H
HO
H
H
H
H
H
H
HO
H
H
H
HO
H
H
HO
H
Cholesterol
M.A. Ott
Pathways and Molecules
Metabolic disorders
•
Inability to synthesize compounds:
Absence of important compounds
•
Inability to degrade compounds:
Accumulation of undesired waste products
E.g., in phenylketonuria
M.A. Ott
Pathways and Molecules
Phenylketonuria (error in phenylalanine catabolism)
O
O
BH4
BH2
O
O
N
O2
Phenylalanine
H2O
N
O
Tyrosine
2-Oxoglutarate
Glutamate
O
O
NADPH,
H+
NADP+
O
O
Phenylpyruvate
O
O
Phenyllactate
M.A. Ott
”Creating the right chemistry” in Pathways
The BioMeta Database
M.A. Ott
”Creating the right chemistry” in Pathways
The BioMeta Database
M.A. Ott
”Creating the right chemistry” in Pathways
BioMeta database structure
M.A. Ott
”Creating the right chemistry” in Pathways
BioMeta database structure
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_7
Molecules
# CASREG
1 1065-31-7
2 387-49-3
3
76-55-1
4 182-20-9
5 450-16-5
6 2754-97-2
7 233-04-8
Reactions
#
1
2
3
Revers
Yes
No
No
R-M Links
R# M#
Role
Stoich
1 1 Substrate
1
1 2 Substrate
1
1 3 Product
1
1 4 Product
1
2 2 Substrate
1
2 3 Substrate
1
2 5 Product
1
2 6 Product
1
3 6 Substrate
2
3 7 Product
1
M.A. Ott
”Creating the right chemistry” in Pathways
Sample database query
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
O
O
EC 1.2.3.4
HO
O
OH
+
O
O
O2
H2O2
O
M.A. Ott
”Creating the right chemistry” in Pathways
Molecular structure identification
• Mesomerism (resonance forms)
R
NH
R
NH2
H2N
+
H2N
+
NH
R
NH
+
NH2
H2N
NH2
• Tautomerism (variable positions of hydrogens)
O
OH
N
HO
HN
N
N
O
OH
NH
N
H
O
• Other “chemical” flexibility
HO
O
HO
OH
OH
OH
• Protonation state
HO
O
HO
OH
OH
OH
M.A. Ott
”Creating the right chemistry” in Pathways
Molecular structures and reactions
O
O
H2N
N
HN
N
O
Carbon imbalance
O
N
OH
HO
No stereochemistry
+
OH Direction
O
H
N
OH
HO P O
P
HO
O
OH
OH
+
HO
NH
N
H
OH
OH
OH
OH
OH
Phosphorus imbalance
OH
HO P O
OH
O No stereochemistry
Reactions:
balancing atoms & charges
adding direction/reversibility
Molecules:
adding stereochemical configurations
“canonicalizing” tautomeric form
O
M.A. Ott
”Creating the right chemistry” in Pathways
And now what’s really going on…
HO
O
OH
HO
HO
O
O
P
O
OH
O
N
O
N
OH
+
HO
stereo
OH
N
O
OH
P
OH
O OH
HO
stereo
2 H2O
N
HO
O
OH
+
N
HN
O
N
H
new
direction
irrevers.
O
O
OH
P
O OH
O OH
HN
HO
+
O
OH
OH
+
HO
P OH
OH
balance
M.A. Ott
”Creating the right chemistry” in Pathways
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
M.A. Ott
”Creating the right chemistry” in Pathways
Validation results
Type of Error
Structure missing
Valence violation(s)
(structures)
# in KEGG
# in BioMeta
# Corrected
1239
1106
133
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
554
366
188
Incorrect stereochemistry
unknown
unknown
69
Total (stereochemistry)
3990
2907
Undefined sp3 stereochemistry
Total corrected
1152
1468
M.A. Ott
”Creating the right chemistry” in Pathways
Validation results
Count
Total
6089
(reaction balance)
Perc.
100.00
Difference
[H+]
Count
*
801
2[H+] *
35
H2O
57
Unknown
766
12.58
Balanced
3711
60.95
O
98
Unbalanced
1612
26.47
H2
17
H2 – 2[H+]
41
2H2
17
H2 – [H+]
18
CH2
30
O2 – H2O
17
Corrected *
893
14.66
*
M.A. Ott
”Creating the right chemistry” in Pathways
Some unresolved issues
O
OH
P
D-Ribose 5-phosphate
O
HO
?
HO
O
OH
P
HO
O
OH
O
O
OH
OH
OH
OH
O
P
P
O
HO
HO
OH
O
OH
OH
HO
O
O
OH
O
OH
HO
OH
2,5-Diamino-6-hydroxy-4-(5'-phosphoribosylamino)-pyrimidine ?
O
OH
P
HO
O
H
N
H2N
O
OH
P
O
N
HO
O
NH
OH
HO
O
H2N
OH
O
N
HO
NH2
H
N
N
OH
NH2
M.A. Ott
”Creating the right chemistry” in Pathways
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
M.A. Ott
”Creating the right chemistry” in Pathways
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
M.A. Ott
”Creating the right chemistry” in Pathways
BioMeta: things to do
• Atom-to-atom correspondence (reaction mapping)
• Tautomer handling
• Pathway searches
• Putative/hypothetical reactions for predicting
metabolism
M.A. Ott
”Creating the right chemistry” in Pathways
Acknowledgements
Felix van Diggelen
Gert Vriend
CMBI