A full mechanistic description of an adaptive landscape requires the maps that describe the transformations of genotype into phenotype and of phenotype into fitness. The genotype-fitness map is the sum of these two component maps.
Dean and colleagues have characterized all three maps controlling coenzyme use in isopropylmalate dehydrogenase (IMDH
), an ancient enzyme with a key function in leucine biosynthesis84
. All IMDHs use the coenzyme NAD+
as an electron acceptor (). By contrast, a highly divergent paralogous enzyme — isocitrate dehydrogenase (IDH
), which catalyses a step in the Krebs’ cycle — uses NAD+
as a coenzyme in some species and NADP+
. To understand why all IMDHs use NAD+
, Dean and colleagues characterized the adaptive landscape on which coenzyme preference evolved.
Constraint and opportunity in the evolution of coenzyme use by paralogous dehydrogenases
The first step was to identify the residues controlling coenzyme use. Guided by crystal structures86,87
, directed mutagenesis was used to replace six residues in the coenzyme-binding pocket of E. coli
IMDH,which uses NAD+
, with the homologus residues from NADP+
using IDHs. Enzyme activities determined in vitro
revealed a dramatic switch in preference, from 200-fold in favour of NAD+
to 200-fold in favour of NADP+
The second step was to characterize the adaptive terrain. Ninety mutants carrying various combinations of residues at the six key sites were constructed, their kinetic activities assayed in vitro
and their fitnesses determined using chemostat competition assays84
. These data were then used to construct the three maps of the adaptive landscape. The genotype-phenotype map of IMDH, which describes the effects that amino-acid replacements have on enzyme activities, shows no epistasis. A simple linear additive model accurately predicts enzyme performance from genotype: adding more residues from NADP+
-dependent IDH always increased the preference for NADP+
. By contrast, the genotype-fitness map of IMDH shows extensive epistasis, with only a few adaptive walks that lead continuously upwards to the adaptive peak.
Chemostat competition assay
A precise assay of the relative growth rates (fitnesses) of competing strains can be obtained in the chemostat, a continuous culture device that is used to impose starvation for a specific resource in a constant environment.
Why the difference in epistasis between the two maps? The answer lies in the concave relationship between phenotype and fitness (). As NAD+ performance increases along the right axis, fitness rises steeply, but soon reaches a plateau where further increases in enzyme activity confer little or no benefit. A similar relationship between NADP+ performance and fitness along the left axis leads to a lower fitness plateau. Epistasis occurs because a mutation that improves the performance of an inefficient enzyme increases fitness substantially, but the same improvement in an already-efficient enzyme produces a negligible increase in fitness.
Why is NADP+
use selected against? The metabolic model underlying the IMDH landscape indicates that the product NADPH, which is abundant in cells, strongly inhibits NADP+
-using IMDHs, reducing their in vivo
activity sufficiently to lower their fitness plateau88
. The NAD+
-using wild type retains high fitness because NADH is scarce in cells, and therefore product inhibition is weak. A directed evolution experiment showed that mutations that weaken NADP+
binding also weaken NADPH binding, relieving the inhibition and increasing fitness88
A library of random mutants that have been generated by PCR amplification of a gene is ligated into a plasmid, transformed into a strain and screened for a desired function.
These findings raised another question: if NADP+
use is deleterious in IMDHs, why do some IDHs use it as a coenzyme? Structural differences in the Michaelis complex of IDH reduce affinity for NADPH, allowing the enzyme to use NADP+
efficiently without intense product inhibition (). But what forces drove some IDH lineages to evolve NADP+
use from an NAD+
-using ancestor 3 billion years ago? Cells use NADH to generate energy in the form of ATP and NADPH to provide reducing power for biosynthesis. Dean and colleagues suggested that the switch in coenzyme might have been an adaptation to growth on oxidized carbon sources such as acetate89
. Consistent with this hypothesis, a genomic association study showed that all 97 sequenced genomes encoding isocitrate lyase (an enzyme that is essential for growth on acetate) have an NADP+
-using IDH, whereas all 32 encoding only NAD+
-using IDHs lack the lyase85
. Competition experiments between E. coli
strains carrying the NADP+
-using wild-type IDH and an engineered NAD+
showed that NADP+
use is indeed advantageous during competition for limiting acetate, but not during competition for limiting glucose89
. Although these experiments confirm that NADP+
use by IDH is advantageous during growth on acetate, they do not resolve whether its historical emergence was a direct adaptation to growth on acetate. Prokaryotes use — and probably used during their early evolution — various oxidized carbon sources.
A complex of substrate bound to enzyme just before catalysis.
Almost three decades ago, Gould and Lewontin drove home the point that the presence of a phenotype does not mean that it was selected for; each hypothesis of adaptation must be tested by proposing a specific mechanism by which the phenotype increases fitness and then determining whether it actually does so through that mechanism91
. Dean and colleagues’ work shows how manipulative techniques can be used to carry out this programme at the molecular level, definitively determining whether specific aspects of a protein’s phenotype are indeed adaptations — and if so, why.