Transcript Evolution & organisation of metabolic Pathways

Evolution & organisation of
metabolic pathways
Bas Kooijman
Dynamic Energy Budget
theory for
metabolic organisation
adult
Dept of Theoretical Biology
Vrije Universiteit, Amsterdam
http://www.bio.vu.nl/thb/deb/
Amsterdam, 2004/03/31
the dynamic structure of life
Central Metabolism
source
polymers
monomers
waste/source
Modules of central metabolism
• Pentose Phosphate (PP) cycle
glucose-6-P
ribulose-6-P,
NADP
NADPH
• Glycolysis
glucose-6-P
pyruvate
ADP + P
ATP
• TriCarboxcyl Acid (TCA) cycle
pyruvate
CO2
NADP
NADPH
• Respiratory chain
NADPH + O2
NADP + H2O
ADP + P
ATP
Evolution of central metabolism
in prokaryotes (= bacteria)
3.8 Ga
2.7 Ga
i = inverse
ACS = acetyl-CoA Synthase pathwayRC = Respiratory Chain Kooijman, Hengeveld 2003
The symbiontic nature of
PP = Pentose Phosphate cycle
Gly = Glycolysis
metabolic evolution
TCA = TriCarboxylic Acid cycle
Acta Biotheoretica (to appear)
Prokaryotic metabolic evolution
Heterotrophy:
• pentose phosph cycle
• glycolysis
• respiration chain
Phototrophy:
• el. transport chain
• PS I & PS II
• Calvin cycle
Chemolithotrophy
• acetyl-CoA pathway
• inverse TCA cycle
• inverse glycolysis
Early ATP generation
FeS2
FeS
H2
S0
H2S
2eS0
H2S
2H2O
2OH-
2H+
ADP
Pi
FeS + S0  FeS2
ADP + Pi  ATP
• ATPase
• hydrogenase
• S-reductase
ATP
2H+
Madigan et al 1997
Substrate processing
Fractions of SU
·· unbound
A· SU-A complex
·B SU-B complex
AB SU-A,B complex
Synthesizing Units:
generalized enzymes
process arriving fluxes
of substrate
reversed flux is small
mixtures of processing
schemes are possible
Kooijman, 2001
Biomass: reserve(s) + structure(s)
Reserve(s), structure(s): generalized compounds,
mixtures of proteins, lipids, carbohydrates: fixed composition
Reserve(s) do complicate model & implications & testing
Reasons to delineate reserve, distinct from structure
• metabolic memory
• biomass composition depends on growth rate
• explanation of
respiration patterns (freshly laid eggs don’t respire)
method of indirect calorimetry
fluxes are linear sums of assimilation, dissipation and growth
inter-species body size scaling relationships
• fate of metabolites (e.g. conversion into energy vs buiding blocks)
Reserve vs structure
Reserve does not mean: “set apart for later use”
compounds in reserve can have active functions
Life span of compounds in
• reserve: limited due to turnover of reserve
all reserve compounds have the same mean life span
• structure: controlled by somatic maintenance
structure compounds can differ in mean life span
Important difference between reserve and structure:
no maintenance costs for reserve
Empirical evidence:
freshly laid eggs consist of reserve and do not respire
Homeostasis
Homeostasis: constant body composition in varying environments
Strong homeostasis  generalized compounds
applies to reserve(s) and structure(s) separately
Weak homeostasis:
ratio reserve/structure becomes and remains constant
if food or substrate is constant (while the individual is growing)
applies to juvenile and adult stages, not to embryos
Implication: stoichiometric constraints on growth
Methanotrophs
CO2
reserve
NH3
CH4
O2
Macro-chemical reaction at fixed growth rate
CH 4  YCX CO2  YNX NH 3 
(1  YCX ) CH nHW OnOW N nNW  YHX H 2O
DEB decomposition into
• assimilation (substrate  reserve)
catabolic & anabolic aspect
• maintenance (reserve  products)
• growth (reserve  structure)
catabolic & anabolic aspect
yield coefficients vary with growth
reserve, structure differ in composition
composition of biomass varies with growth
Kooijman, Andersen & Kooi 2004
Anammox
Macro-chemical reaction at r = 0.0014 h-1
1 NH 4  1.32 NO2  0.068 HCO3  0.128 H  
1.025 N 2  0.260 NO3  0.068 CH 2O0.5 N 0.15  2.030 H 2O
DEB decomposition into
• assimilation (substrate  reserve)
catabolic & anabolic aspect
• maintenance (reserve  products)
• growth (reserve  structure)
catabolic & anabolic aspect
yield coefficients vary with growth
reserve, structure differ in composition
composition of biomass varies with growth
rm = 0.003 h-1; kE = 0.0127 h-1; kM = 0.0008 h-1
ySE = 8.8; yVE = 0.8
nHE = 2; nOE = 0.46; nNE = 0.25
nHV = 2; nOV = 0.51; nNV = 0.125
Brandt, 2002
Nitrogen cycle
Brocadia anammoxidans
some cyanobacteria,
Azotobacter, Azospirillum,
Azorhizobium, Klebsiella,
Rhizobium,some others
Nitrosomonas
Some crucial conversions
depend on few species
Nitrobacter
many
CHON= biomass
Syntrophy
Coupling hydrogen & methane production
energy generation aspect at aerobic/anaerobic interface
ethanol
acetate

3

dihydrogen
2 C2 H 6O  2 H 2O  O2  2 C2 H 3O  2 H  4 H 2
dihydrogen bicarbonate

3

methane
4 H 2  CHO  H  CH 4  3 H 2O
Total: 2 C2 H 6O  CO2  O2  CH 4  2 C2 H 3O3  2 H 
methane hydrates
>300 m deep, < 8C
linked with nutrient supply
Product Formation
According to
Dynamic Energy Budget theory:
pyruvate, mg/l
Product formation rate =
wA . Assimilation rate +
wM . Maintenance rate +
wG . Growth rate
For pyruvate: wG<0
Applies to all products, heat
& non-limiting substrates
Indirect calorimetry (Lavoisier, 1780):
heat = wO JO + wC JC + wN JN
No reserve:
2-dim basis for product formation
throughput rate, h-1
Glucose-limited growth of Saccharomyces
Data from Schatzmann, 1975
Symbiosis
substrate
product
Symbiosis
substrate
substrate
Steps in symbiogenesis
Free-living, homogeneous
Structures merge
Free-living, clustering
Internalization
Reserves merge
biomass density
Chemostat Steady States
Free living
Products substitutable
Free living
Products complementary
Exchange on flux-basis
Structures merged
throughput rate
symbiont
host
Endosymbiosis
Exchange on conc-basis
Reserves merged
Host uses 2 substrates
Symbiogenesis
• symbioses: fundamental organization of life based on syntrophy
ranges from weak to strong interactions; basis of biodiversity
• symbiogenesis: evolution of eukaryotes (mitochondria, plastids)
• DEB model is closed under symbiogenesis:
it is possible to model symbiogenesis of two initially independently
living populations that follow the DEB rules by incremental changes
of parameter values such that a single population emerges that
again follows the DEB rules
• essential property for models that apply to all organisms
Kooijman, Auger, Poggiale, Kooi 2003
Quantitative steps in symbiogenesis and the evolution of homeostasis
Biological Reviews 78: 435 - 463
Symbiogenesis
1.5-2 Ga
1.2 Ga
Eukaryote metabolic evolution
First eukaryotes: heterotrophs by symbiogenesis
compartmental cellular organisation
Acquisition of phototrophy
frequently did not result in loss of heterotrophy
Acquisition of membrane transport
between internalization of mitochondria and plastids
No phagocytosis in fungi & plants; loss?
pinocytosis in animals = phagocytosis in e.g. amoeba?
Direct link between phagocytosis and membrane transport?
Membrane traffic
The golgi apparatus serves
as a central clearing house
and channel between the
endo- and exoplasmic domains
1 ER-Golgi shuttle
2 secretory shuttle between
Golgi and plasma membrane
2’ crinophagic diversion
3 Golgi-lysosome shuttle
3’ alternative route from Golgi
to lyosomes via the plasma
membrane and an endosome
4 endocytic shuttle between the
plasma membrane and an
endosome
4’ alternative endocytic pathway
bypassing an endosome
5 plasma membrane retrieval
6 endosome-lysosome pathway
7 autophagic segregation
From: Duve, C. de 1984 A guided tour of the living cell, Sci. Am. Lib., New York
Clathrin unknown in prokaryotes
Chloroplast dynamics
Coordinated movement of chloroplasts through cells
Sizes of blobs
do not reflect
number of species
Survey of organisms
Myxomycota
Protostelida
Bikont
DHFR-TS gene fusion
loss phagoc.Apusozoa
membr. dyn
unikont
mainly celllose
gap junctions
tissues (nervous)
mitochondria
bicentriolar
primary
mainly chitin
chloroplast
EF1 insertion
secondary
Plasmodiophoromycota
Chlorarachnida
Cercozoa
Cercomonada
chloroplast Amoebozoa
Archamoeba
tertiary
chloroplast
photo
symbionts
Bacteria
Bacteria
Rhizopoda
Sporozoa
Percolozoa
Excavates
Euglenozoa
Loukozoa
AlveoDinozoa
lates
Ciliophora
chloroplasts
Chytridiomycota
cortical alveoli
Actinopoda
(brown algae)
Phaeophyceae
Xanthophyceae
Raphidophyceae
Chrysophyceae
Synurophyceae
Eustigmatophyceae
Labyrinthulomycota
Dictyochophyceae
Bicosoecia
Pedinellophyceae
Pelagophyceae
Bigyromonada
Bacillariophyceae
Pseudofungi
(diatoms)
Bolidophyceae
Opalinata
Prymnesiophyceae
Metamonada
Cryptophyceae
triple roots
Granuloreticulata
forams
Xenophyophora
Basidiomycota
Ascomycota
fungi
Glomeromycota
Zygomycota
Microsporidia
animals
animals
Choanozoa
Composed by
Bas Kooijman
(plants)
Cormophyta
(green algae)
Chlorophyceae
Plantae
(red algae)
Rhodophyceae
Glaucophyceae
Cells, individuals, colonies
vague boundaries
• plasmodesmata connect cytoplasm; cells form a symplast: plants
• pits and large pores connect cytoplasm: fungi, rhodophytes
• multinucleated cells occur; individuals can be unicellular:
fungi, Eumycetozoa, Myxozoa, ciliates, Xenophyophores, Actinophryids, Biomyxa, diplomonads,
Gymnosphaerida, haplosporids, Microsporidia, nephridiophagids, Nucleariidae, plasmodiophorids,
Pseudospora, Xanthophyta (e.g. Vaucheria), most classes of Chlorophyta (Chlorophyceae, Ulvoph
Charophyceae (in mature cells) and all Cladophoryceae, Bryopsidophyceae and Dasycladophycea
• cells inside cells: Paramyxea
• uni- and multicellular stages: multicellular spores in unicellular myxozoa, gametes
• individuals can remain connected after vegetative propagation: plants, corals, b
• individuals in colonies can strongly interact
and specialize for particular tasks:
syphonophorans, insects, mole rats
Kooijman, Hengeveld 2003
The symbiontic nature of
metabolic evolution
Acta Biotheoretica (to appear)
Heterocephalus glaber
rotifer
Conochilus
hippocrepis
(Endo)symbiosis
Frequent association between photo- and heterotroph
photo  hetero: carbohydrates (energy supply)
photo  hetero: nutrients (frequently NH3 or NO3-)
most (perhaps all) plants have myccorrhizas,
the symbiosis combines photolithotrophy and organochemotrophy
Also frequent: association between phototroph and N2-fixer
where N2-fixer plays role of heterotroph
Symbiosis: living together in interaction (basic form of life)
Mutualism: “benefit” for both partners
symbioses need not be mutualistic
“benefit” frequently difficult to judge and anthropocentric
Syntrophy: one lives of products of another (e.g. faeces)
can be bilateral; frequent basis of symbiosis
Chlorochromatium (Chlorobibacteria, Sphingobacteria)
(= Chlorochromatium)
From: Margulis, L & Schwartz, K.V.
1998 Five kingdoms.Freeman, NY
(Endo)symbiosis
Paramecium bursaria
ciliate with green algae
Cladonia diversa
ascomycete with green algae
Ophrydium versatile
ciliate with green algae
Peltigera
ascomycete with green algae
(Endo)symbiosis
Chlorophyte symbionts
visible through microscope
Grazed by reindeer in winter
Rangifer tarandus
Lichen Cladonia portentosa
Mitochondria
TriCarboxylic Acid cycle (= Krebs cycle)
Enzymes pass metabolites directly to other enzymes
enzymes catalizing transformations 5 & 7:
bound to inner membrane (and FAD/FADH2)
Net transformation:
Acetyl-CoA + 3 NAD+ + FAD + GDP 3- + Pi2- + 2 H2O =
2 CO2 + 3 NADH + FADH2 + GTP 4- + 2 H+ + HS-CoA
Dual function of intermediary metabolites
building blocks  energy substrate
Transformations:
1 Oxaloacetate + Acetyl CoA + H2O = Citrate + HSCoA
2 Citrate = cis-Aconitrate + H2O
3 cis-Aconitrate + H2O = Isocitrate
4 Isocitrate + NAD+ = α-Ketoglutarate + CO2 + NADH + H+
5 α-Ketoglutarate + NAD+ + HSCoA = Succinyl CoA + CO2 + NADH + H+
6 Succinyl CoA + GDP 3- + Pi 2- + H+ = Succinate + GTP 4- + HSCoA
7 Succinate + FAD = Fumarate + FADH2
8 Fumarate + H2O = Malate
9 Malate + NAD+ = Oxaloacetate + NADH + H+
all eukaryotes
once possessed
mitochondria,
most still do
enzymes are located
in metabolon;
channeling of
metabolites
Pathways & allocation
structure
structure
maintenance
reserve
maintenance
reserve
structure
maintenance
reserve
Mixture of products &
intermediary metabolites
that is allocated to
maintenance (or growth)
has constant composition
Kooijman & Segel, 2004
Numerical matching for n=4
0
3
4
4
3
2
Unbound fraction
Product flux
1
2
1
Spec growth rate
Rejected flux
0
1
2
3
Spec growth rate
 = 0.73, 0.67, 0.001, 0.27 handshaking
 = 0.67, 0.91, 0.96, 0.97 binding prob
k = 0.12, 0.19, 0.54, 0.19 dissociation
nSE = 0.032,0.032,0.032,0.032 # in reserve
nSV = 0.045,0.045,0.045,0.045 # in structure
yEV = 1.2 res/struct kE = 0.4 res turnover
jEM = 0.02 maint flux n0E = 0.05 sub in res
Matching pathway  whole cell
No exact match possible between
production of products and intermediary metabolites by pathway
and requirements by the cell
But very close approximation is possible by tuning
abundance parameters nSi E , nSiV
and/or
binding and handshaking parameters ρi , αi
Good approximation requires all four tuning parameters per node
growth-dependent reserve abundance plays a key role in tuning
Kooijman, S. A. L. M. and Segel, L. A. (2004)
How growth affects the fate of cellular substrates.
Bull. Math. Biol. (to appear)