FEATURE ARTICLE
The Molecular Anatomy of an Ancient Adaptive Event
Protein engineering identifies the structural basis of a 3.5 billion-year-old adaptation
Antony Dean
An Adaptive Event: When and Why
Mitochondria are the powerhouses of eukaryotic cells, where ATP is synthesized. Their ancestors were free-living eubacteria, until they were engulfed by a primitive eukaryotic cell some 3.5 billion years ago. Although most mitochondrial genes were lost long ago, a few, including those of the mitochondrial ICDH, were transferred onto the chromosomes in the eukaryotic nucleus. So, like modern eubacterial ICDHs, mitochondrial ICDHs are the direct descendants of an ancient eubacterial ICDH. However, unlike the majority of modern eubacterial ICDHs, which are NADP-dependent, all mitochondrial ICDHs are NAD-dependent. This immediately suggests that a switch in coenzyme usage took place very early in the history of life: Either the ancestral eubacterial ICDH used NADP, and NAD use by modern mitochondrial ICDHs is a derived trait, or the ancestral eubacterial ICDH used NAD, and NADP use by modern eubacterial ICDHs is derivative.
Any attempt to distinguish between these two competing possibilities lies firmly in the domain of phylogenetics, a field that seeks to reconstruct the genealogical relationships among organisms. The results of a phylogenetic analysis are usually presented in the form of a tree. The process begins by aligning the sequence of amino acids in a protein such that the amino acid positions in one ICDH correspond to those in another. Next, the molecular phylogenetic tree is constructed. The third step is to identify the root of the tree—the one position in the tree from which all sequences ultimately arose. And the fourth and final step is to interpret the tree in an evolutionary context.
Most sequence alignments are based on some algorithmic criterion, such as maximizing the number of identical amino acids among the proteins. This works well for sequences that are similar, such as the eubacterial ICDHs or the ICDHs found in the eukaryotic cytoplasm (Figure 1). However, eubacterial ICDHs are so very dissimilar in their sequences from eukaryotic cytoplasmic ICDHs that little confidence can be placed in the alignments between these groups, no matter how powerful the computer program used.
Fortunately there is another, exquisitely precise though little used, means to align sequences. Figure 1 shows a comparison of the basic frameworks of the ICDH from the bacterium Escherichia coli and the isopropylmalate dehydrogenase, or IPMDH, from the bacterium Thermus thermophilus. IPMDH carries out a similar reaction to ICDH in the biosynthesis of the amino acid leucine. Visual inspection reveals that the frameworks are quite similar. This is quite remarkable when one considers that only 20 percent of their amino acids are shared in common. On closer inspection many structural features, such as the flat sheets and the helices on the surfaces, obviously superimpose. There are some differences between these structures, though. For example, ICDH possesses a helix and a loop for which there is no equivalent in IPMDH. Here, a gap must be introduced into the amino acid sequence of IPMDH when aligning it against that of ICDH.
Enzymes catalyze reactions by binding and aligning the reagents—substrates and coenzymes—at specific sites on their surfaces called active sites (Figure 6). The specific amino acids (called amino acid residues when in a protein) crucial to the binding of both substrate and coenzyme and to catalysis in ICDH have been identified using x-ray crystallography (Figure 7). Being essential for function, these residues are necessarily highly conserved in the course of evolution. They provide key landmarks when aligning sequences from other distantly related groups, such as the mitochondrial and cytoplasmic ICDHs of eukaryotes, for which x-ray structures are not yet available. A knowledge of the three-dimensional structures of just two proteins, E. coli ICDH and T. thermophilus IPMDH, proved essential to the successful completion of the first step—obtaining a reliable alignment for this entire family of dehydrogenases.
The second step is to construct the phylogeny. There are a number of approaches to do this, each with its own adherents, even vehement supporters. Pheneticists construct phylogenetic trees based on the percentage of similarities between pairs of amino acid sequences. As an example, take the four amino acid sequences AKGCV, ALMSD, CLMQS and CGLCV, in which letters designate particular amino acid residues. These sequences can be grouped into two pairs, the first with the last and the second with the third, on the basis that sequences within a pair differ at three sites only, whereas sequences drawn from different pairs differ at a minimum of four sites. Thus, the first and last sequences resemble each other more closely than they resemble the second and third, which are deemed to form a distantly related group.
Cladists take a fundamentally different and perhaps a less intuitive approach, attempting to reconstruct the topology (branching order) of a tree using only shared differences. Of the five positions in these sequences only the first would be used by a cladist because the first two sequences "share" an A and the second two sequences "share" a C. In pairing the first sequence with the second and the third with the fourth, a cladist arrives at a rather different phylogeny than the pheneticist. Different methods sometimes yield different phylogenies.
So which is the most reliable method for reconstructing phylogenies? The answer is unambiguous: We biologists do not know. So, rather than getting bogged down in the perennial debate as to which of the various methods is best, we use a variety of approaches (the phenetic techniques of neighbor joining and maximum likelihood and the cladistic technique of maximum parsimony) to recover phylogenetic trees that, within statistical error, are indistinguishable. All these trees consist of four distantly related groups of enzymes (Figure 8): the eubacterial NADP-dependent ICDHs, the eukaryotic mitochondrial NAD-dependent ICDHs, the eukaryotic cytoplasmic NADP-dependent ICDHs and the eubacterial and eukaryotic NAD-dependent IPMDHs. The four groups join at a central node. The precise pairwise branching order at this node is not resolved in these data.
As it stands, this tree is unrooted. There is no sequence that we can confidently assert should join the tree at its most ancient origin, the root. But we need a root because a root provides directionality in time: It tells us what is ancient and what is modern. Fortunately, a biochemical argument can be used to place a root on this tree. Ancient single-celled organisms must have synthesized all the amino acids essential for life because they could not have obtained them from diet—when life first began there was literally nothing to eat. They must have had an ICDH activity to synthesize glutamate, and they must have had an IPMDH activity to synthesize leucine. Therefore, the root of the tree lies on the limb joining the ICDHs to the IPMDHs. This completes step three.
The final step is to interpret the rooted tree (Figure 8) in an evolutionary context. An ancient single-celled organism had a single gene encoding a primitive enzyme that had the catalytic activities of both ICDH and IPMDH. The gene duplicated, and further evolution produced the first true ICDH and the first true IPMDH. Later, the eukaryotes emerged, an event reflected in both the ICDH and the IPMDH branches of the tree.
The tree also reveals that NADP usage in the ICDHs may have evolved independently twice, once in the eukaryote lineage and once in the eubacterial lineage. Both events took place around the time that bacteria were first taken up by primitive eukaryotes, which led to the modern mitochondria—some 3.5 billion years ago judged from fossil evidence. Thus, the order in which coenzyme usage evolved is resolved in favor of NAD dependence being ancestral to NADP dependence.
But why did an ancient bacterial NAD-dependent ICDH evolve the ability to use NADP instead? As I have shown, such a switch represents a major shift in metabolic role, from energy production to biosynthesis. Adaptation to using acetate as the sole source of carbon and energy provides a possible explanation for the evolution of NADP dependence. All modern bacteria capable of growing on acetate generate the vast quantities of NADPH needed for biosynthesis in the citric acid cycle (Figure 9). E. coli, for example, uses ICDH to generate 90 percent of the NADPH necessary for growth on acetate. In contrast, bacteria retaining an NAD-dependent ICDH are incapable of growing on acetate. The switch by ICDH in coenzyme usage, from NAD to NADP, represents an adaptation, one of several in fact, to growth on acetate as the sole source of carbon and energy.
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