Old Enzymes Tell Stories
by Elio
Offhand, what can you say about the enzymes of the earliest cells? Surely, a few things pop up, for example, they were fewer in number and probably simpler than the ones we have now. They must have had fewer frills than the modern ones, which tend to have clear-cut tastes for substrates and are very efficient in catalyzing reactions. Ancient enzymes were likely to be more promiscuous in their activities, which suggests that ancestral cells could make do with a small number of them. However, these enzymes were likely good at multitasking, catalyzing distinct reactions, which is in keeping with the notion that early cells had small genomes. This idea is referred to as the Yčas/Jensen model, after the two people who proposed it independently in 1974 and 1976, respectively. In time, evolution took care of making larger genomes, allowing enzymes to become more varied as well as more specific. The fact that many modern enzymes have multiple activities may reflect the persistence of vestigial properties of the older enzymes. And one or more of such vestigial properties may be, in many cases, no longer physiologically relevant today. Using such thinking, phylogenic approaches have helped to resurrect what may be ancestral enzymes (see here and here).
Here I will deal with two papers on the topic of ancient enzymes, each with its own trajectory. The first one tries to substantiate the point made above, that the early enzymes carried out more than one activity that was physiologically pertinent back then. Eventually, these activities were taken over by separate, more specific enzymes. The authors of this paper reason that the existence of enzymes that resemble the ancient ones may be best revealed in cells with reduced genomes. The second paper claims what may superficially seem like the converse, that ancient enzymes were limited in their catalytic repertoire. However, these authors proposed that this limitation is solely in the ability of these enzymes to function in viral replication. On reflection, the two are separate arguments, thus both may be right.
In Search of Primitive Enzymes
A New Zealand group led by Wayne Patrick examined in some detail modern enzymes with multiple activities one of which is likely to be a relic. The authors define the enzymes of interest here as having a single active site that carries out more than one catalytic reaction. One such known enzyme is E. coli 's cystathionine beta-lyase (CBL), which, in addition to being responsible for a late step in methionine biosynthesis (making homocysteine), also has alanine racemase activity (the interconversion of L- into the D‑alanine required for peptidoglycan biosynthesis). However, in E. coli, the physiologically relevant racemase activity is not carried out by CBL but by a different enzyme, alanine racemase (ALR), suggesting that the racemase activity of CBL is vestigial and probably physiologically immaterial (Figure 1).
Figure 1. Structure and function of CBL and ALR. A The CBL tetramer, which adopts fold type I. The β‑elimination reaction catalyzed by CBL in methionine biosynthesis is shown below the structure. B The fold type III ALR and the reaction it catalyzes to provide D‑alanine for peptidoglycan biosynthesis. In each structure, the PLP cofactors are shown in space-filling format. © The American Society for Biochemistry and Molecular Biology
If you're still with me, you will recognize that CBL is a good candidate for a primordial-like enzyme such as predicted by the model. If so, do any contemporary bacteria carry out two biochemical activities with a single enzyme like CBL? Examining a bunch of genomes, they found the genes for such an enzyme in only three species, all with reduced genomes: Pelagibacter ubique, a Wolbachia endosymbiont of drosophila, and Thermotoga maritima. They looked in these strains for possible genuine ALRs but found none, meaning that the resident CBL can be assumed to be the one that carries out the racemase function. The CBL of these three bacteria then represents the kind of primordial bifunctional enzymes the investigators were looking for. And not only on paper! At 37°C, expression of each enzyme rescued an alanine racemase knockout strain, E. coli MB2795, as quickly as expressing E. coli ALR itself.
Given that so many enzymes are in fact multifunctional, the authors propose that examples of primordial-like enzymes are abundant. In their words: 'We… suggest that esoteric enzymes – such as the poorly-active multifunctional ones we have characterized in this study – represent the rule, and not the exception, in the biosphere.' The search should be straightforward: consider enzyme X that has a second minor activity that in most species is carried out primarily by enzyme Y and look among cells with reduced-genome for enzyme X where Y is absent. In such cells, enzyme X should qualify as a potential primordial type that may have been present in early cells. Sounds a bit like using synthetic lethal technology.
Can Ancestral Proteins Keep Viruses From Replicating?
Figure 2. Graphical representation of sequence identity of thioredoxins and efficiency of phage propagation. Source
Let's now jump into the second question. Primitive enzymes, multifunctional though they may have been, are not likely to be functional in viral multiplication (many phages use host enzymes for their transactions). Viral high-jacking of host functions must have evolved quite a bit later, perhaps along with the viruses themselves. This is the subject of a recent paper by a highly experienced group from the University of Granada, Spain led by José Sanchez-Ruiz. They argue that substituting a modern enzyme ‒ one that works for both host and virus ‒ with one that works for the host only should inhibit viral development.
The proof of concept relies on experiments with thioredoxins, small redox enzymes that are probably found in all organisms, where they perform a myriad of functions. In bacteria, they participate in transcriptional regulation, energy transduction, and oxidative stress response, among others. Of interest here is that thioredoxin functions in DNA replication, both bacterial and viral. In coliphage T7, thioredoxin works as a 'processivity factor' that increases the number of nucleotides traversed by the replication complex from a few bases to several hundred bases per binding event. Now a bit of biochemistry: thioredoxin binds with great affinity to T7 DNA polymerase via a 78-amino-acid fragment known as the thioredoxin-binding domain. This binding apparently changes the conformation of the DNA polymerase to enhance its docking on the DNA and keeping the replication complex from hopping on and off.
Figure 3. Schematic phylogenetic tree showing the precambrian phylogenetic nodes targeted in this work and their geological ages. The nodes targeted for reconstruction are: LBCA (last bacterial common ancestor), LPBCA (last common ancestor of Cyanobacteria, Deinococcus, and Thermus groups), LGPCA (the last common ancestor of γ‑proteobacteria), AECA (archaeal-eukaryotic common ancestor), LACA (last archaeal common ancestor), LECA (last eukaryotic common ancestor), and LAFCA (last common ancestor of fungi and animals). The numbers alongside the nodes stand for the values of the identity of the reconstructed sequences with the sequence of E. coli thioredoxin 1. Source
We're getting to the point. The authors proposed using ancestral proteins to test the prediction that these would work with E. coli but not with phages. You obviously cannot order such enzymes from your supply house, but they can be resurrected using phylogenetic information. Reconstructing ancient proteins has become a fun field of study of its own (see a review here). In fact, ancestral thioredoxins that represent very ancient forms (Precambrian) have been prepared and studied. They are both active and extra-thermostable. How did the authors perform such a revival? In brief, they undertook extensive phylogenetic analyses and, from their position on evolutionary trees, they selected the genes likely to have encoded old enzymes (Figure 3). The next steps are straightforward: amplify the appropriate fragments using PCR, clone them into a suitable vector, express the proteins in E. coli, determine their properties.
So, what did they find? First, E. coli can grow without its two thioredoxins (yes, it has two), but at a slower rate (how I wish the authors had plotted their growth data on semi-log paper, which allows one to compare growth rates directly). Introducing genes for ancestral thioredoxins restored the faster growth phenotype. They tried a number of candidates for growth restoration (Figures 2 + 4). The enzyme that was best at it was one derived from the last common ancestor of Cyanobacteria and the Deinococcus-Thermus branch, bacteria that are likely to have lived in ancient high-temperature environments (the 'resurrected' thioredoxin denatures at temeratures >123°C). Now for the key experiment, testing the growth of phage T7 in these strains. Sure enough, the phage did not grow. They say: 'The implication is that ancestral thioredoxins that show substantial redox functionality within E. coli (as determined by their capability to rescue the slower-growth phenotype of an E. coli trx‒ strain) do not allow (or substantially limit) phage propagation in E. coli.' They further say: 'In the specific case studied here, we had to "travel back in time" to 2.5 billion years ago… to find an ancestral thioredoxin that prevents propagation of bacteriophage T7 in E. coli.'
Figure 4. Effect of precambrian thioredoxins on the propagation of bacteriophage T7. Efficiency of T7 propagation in E. coli complemented with human and ancestral thioredoxins (left panel). Data are given as percentage of the maximal efficiency with E. coli thioredoxin. Generation times at 37°C are displayed in the right panel. Source
Not only are these considerations of specific interest, but they may have applications. Substituting the gene for a native enzyme involved in viral reproduction for the ancestral single-minded one may make a plant or animal resistant to the virus. The authors outline a protocol that may be relevant here. They point out that viruses would have a hard time mutating to the use of ancestral proteins with limited capabilities. Thus, a host that received an analog of an ancient enzyme would become immune to the virus. Such gene substitution may not be feasible throughout, but in some instances, perhaps with plants, they may have practical applications. Worth a try, no?
Isn't it surprising that old enzymes tell stories? And good ones, at that.
We discussed this paper in This Week in Microbiology (TWiM), episode 157.
