Ann Gauger

Editor’s note: Nylon is a modern synthetic product used in the manufacturing, most familiarly, of ladies’ stockings but also a range of other goods, from rope to parachutes to auto tires. Nylonase is a popular evolutionary icon, brandished by theistic evolutionist Dennis Venema among others. In a series of three posts, Discovery Institute biologist Ann Gauger takes a closer look.

A significant problem for the neo-Darwinian story is the origin of new biological information. Clearly, information has increased over the course of life’s history — new life forms appeared, requiring new genes, proteins, and other functional information. The question is — how did it happen? This is the central question concerning the origin of living things.

Stephen Meyer and Douglas Axe have made this strong claim:

[T]he neo-Darwinian mechanism — with its reliance on a random mutational search to generate novel gene sequences — is not an adequate mechanism to produce the information necessary for even a single new protein fold, let alone a novel animal form, in available evolutionary deep time.

Their claim is based on the experimental finding by Doug Axe that functional protein folds are exceedingly rare, on the order on 1 in 10 to the 77th power, meaning that all the creatures of the Earth searching for the age of the Earth by random mutation could not find even one medium-size protein fold.

In contrast, Dennis Venema, professor of biology at Trinity Western University, claims in his book Adam and the Genome and in posts at the BioLogos website that getting new information is not hard. In his book, he presents several examples he thinks demonstrate the appearance of new information — the apparent evolution of new protein binding sites, for example. But the best way to reveal Axe and Meyer’s folly, he thinks, (and says so in his book and a post at BioLogos) would be to show that a genuinely “new” protein can evolve.

…[E]ven more convincing… would be an actual example of a functional protein coming into existence from scratch — catching a novel protein forming “in the act” as it were. We know of such an example — the formation of an enzyme that breaks down a man-made chemical.

In the 1970s, scientists made a surprising discovery: a bacterium that can digest nylon, a synthetic chemical not found in nature. These bacteria were living in the wastewater ponds of chemical factories, and they were able to use nylon as their only source of food. Nylon, however, was only about 40 years old at the time — how had these bacteria adapted to this novel chemical in their environment so quickly? Intrigued, the scientists investigated. What they discovered was that the bacteria had an enzyme (which they called “nylonase”) that effectively digested the chemical. This enzyme, interestingly, arose from scratch as an insertion mutation into the coding sequence of another gene. This insertion simultaneously formed a “stop” codon early in the original gene (a codon that tells the ribosome to stop adding amino acids to a protein) and formed a brand new “start” codon in a different reading frame. The new reading frame ran for 392 amino acids before the first “stop” codon, producing a large, novel protein. As in our example above, this new protein was based on different codons due to the frameshift. It was truly “de novo” — a new sequence.

Venema is right. If the nylonase enzyme did evolve from a frameshifted protein, it would genuinely be a demonstration that new proteins are easy to evolve. It would be proof positive that intelligent design advocates are wrong, that it’s not hard to get a new protein from random sequence. But the story bears reexamining. Is the new protein really the product of a frameshift, or did it pre-exist the introduction of nylon into the environment? What exactly do we know about this enzyme? Does the evidence substantiate the claims of Venema and others, or does it lead to other conclusions?

First, some history. In the 1970s Japanese scientists discovered that certain bacteria had developed the ability to degrade the synthetic polymer nylon. Okada et al. identified three enzymes responsible for nylon degradation, and named them EI, EII, and EIII. The genes that encoded them were named nylA,nylB, and nylC. They sequenced the plasmid on which the genes were found, and discovered that there was another gene on the same plasmid that was very similar to nylB; they named it nylB′. (We will focus on the story of nylB and nylB′ because they are the ones relevant to Venema’s story.)

So far all I have given you are the facts. Now here’s the interpretation of these facts. Some claimed that the nylonase enzyme, as it was called, had originated some time after people began making nylon (in the 1930s). That seemed plausible because nylonase was unable to degrade naturally occurring amide bonds — it could degrade only the amide bonds in nylon — and so had not existed previously, it was thought. The popular conclusion was that the nylonase activity evolved in response to the presence of nylon in the environment, and thus was only forty years old. And here’s the big interpretive leap: it must not be hard to get new enzymes if a new one can evolve within a period of forty years.

Okada et al. had sequenced the genes encoding nylB and nylB′. They concluded that the nylonase activity was the result of a gene duplication followed by several mutations to the nylB gene. But at this point Susumu Ohno, an eminent molecular geneticist and evolutionary biologist, noticed something unusual about the nylB gene sequence (Ohno, 1984). Ohno had a theory that DNA with repeats of the right kind had the potential to code for protein in multiple frames, with no interrupting stop codons, and might thus be a source for “new” proteins. (If you are unfamiliar with the terms I just used, I invite you to take a look at my post tomorrow, where I will explain the necessary concepts. For those already familiar, I present some relevant data concerning the rarity of sequences that can be frameshifted.)

Ohno noticed that nylB, the gene for nylonase, might originally have encoded something else if a certain T was removed. The nylonase gene as it exists now has 1179 bases, which encode a 392 amino acid protein. Without a particular T embedded in the ATG start codon, though, the sequence would have specified a hypothetical original gene with a longer open reading frame (ORF) of 427 amino acids, in a different frame. Thus, Ohno proposed a “new” protein with a new function acting on a new substrate was born when a T inserted in between a particular A and G in the DNA, making a new ATG start codon and shifting the frame to code for a new protein, the protein we now call nylonase.

Ingenious. According to Ohno, nylonase could be a new enzyme, appearing suddenly with no known precursors via a sudden frameshift. (Note that all of this assumes that new protein folds are easy to get.) Ohno published this hypothesis in the Proceedings of the National Academy of Sciences. It was a hypothesis only, however, as a careful reading of his paper shows. One heading, for example:

R-IIA Coding Sequence [nylB] for 6-AHA LOH [nylonase] Embodies an Alternative, Longer Open Reading Frame That Might Have Been the Original Coding Sequence [Emphasis added.]

and the text says:

I suggest that the RS-IIA base sequence [nylB] was originally a coding sequence for an arginine-rich polypeptide chain 427 or so residues long in its length and that the coding sequence for one of the two isozymic forms of 6-ALA LOH [nylonase] arose from its alternative open reading frame. [Emphasis added.]

Ohno presented arguments for why his suggestion was plausible, but did not provide evidence that the “original” gene ever existed or was used (in fact he says it was unlikely to be useful based on its amino acid composition), or that the insertion ever happened. Nonetheless, the frame-shift hypothesis for the origin of nylonase has been widely proclaimed as fact (though, notably, not by Okada et al. who have done most of the work).

If the nylonase story as told above were true, namely that a frameshift mutation resulted in the de novo generation of a new protein fold with a new function, it would indeed constitute a substantial refutation to Meyer and Axe’s claim. If a frame-shift mutation can produce a random new open reading frame in real, observable time, and give rise to a new functional enzyme, then it must not be that hard to make new functional protein folds. In other words, functional protein folds must not be rare in sequence space. And therefore Stephen Meyer’s arguments about the difficulty of getting enough new biological information to generate a new fold must be wrong as well. Venema flatly asserts:

If de novo protein-coding genes such as nylonase can come into being from scratch, as it were, then it is demonstrably the case that new protein folds can be formed by evolutionary mechanisms without difficulty….[I]f Meyer had understood de novo gene formation — as we have seen, he mistakenly thought it was an unexplained process — he would have known that new protein folds could indeed be easily developed by evolutionary processes.

Slam dunk, right?

A little caution in accepting this story without hard evidence would be wise. In genetics we are taught that frame-shift mutations are extremely disruptive, completely changing the coding sequence and resulting in truncated nonsense. In fact, one term for a frameshift mutation is “nonsense mutation.” A biologist’s basic intuition should be that frameshifts are highly unlikely to produce something useful. The only reasons for the widespread acceptance of Ohno’s hypothesis that I can come up with are the unusual character of the sequence itself, Ohno reputation as a brilliant scientist (which he was), and wish-fulfillment on the part of some evolutionary biologists.

Fortunately, science marches on, and evidence continues to accumulate. The same group of Japanese scientists continued their study of the nylonase genes. nylB appeared to be the result of a gene duplication of nylB′ that occurred some time ago. EII′ (the enzyme encoded by nylB′) had very little nylonase activity, while EII (the enzyme encoded by nylB) was about 1000 fold higher in activity. The two enzymes differed in amino acid sequence at 47 positions out of 392. With some painstaking work, the Japanese determined that just two mutations were sufficient to convert EII′ to the EII level of activity.

They then obtained the three-dimensional structure of an EII-EII′ hybrid protein. And with those results everything changed — or should have.

Here’s what Venema takes from the paper and interprets the evidence:

…the three-dimensional structure of the protein has been solved using X-ray crystallography, a method that gives us the precise shape of the protein at high resolution. Nylonase is chock full of protein folds— exactly the sort of folds Meyer claims must be the result of design because evolution could not have produced them even with all the time since the origin of life. [Emphasis added.]

Unfortunately, Venema doesn’t have the story straight. Nylonase has a particular fold, a particular three-dimensional, stable shape. Most proteins have a distinct fold — there are several thousand kinds of folds known so far, each with a distinct topology and structure. Folds are typically made up of small secondary structures called alpha helices and beta strands, which help to assemble the tertiary structure — the fold as a whole. Venema seems unclear about what a protein fold is, and the distinction between secondary and tertiary structures. Nylonase is not “chock full of folds.” No structural biologist would describe nylonase as “chock full of protein folds.” Indeed, no protein is “chock full of folds.” Perhaps Venema was referring to the smaller units of secondary structure I mentioned above, the alpha helices or beta strands. But it would appear he doesn’t know what a protein fold is.

Maybe that explains why Venema missed the essential point of the paper describing nylonase’s structure. The crystal structure of EII-EII’ (a nylonase hybrid necessary to be able to crystalize the protein) revealed that it is not a new kind of fold, but a member of the beta-lactamase fold family. More specifically, it resembles carboxylesterases, a subgrouping of that family. In addition, when the scientists checked EII′ and EII, they found that both enzymes had previously undetected carboxylesterase activity. In other words, the EII’ and EII enzymes were carboxylesterases. If it looks like a duck and quacks like a duck, it is a duck.

Thus, EII′ and EII did not have frameshifted new folds. They had pre-existing folds with activity characteristic of their fold type. There was no brand-new protein. No novel protein fold had emerged. And no frameshift mutation was required to produce nylonase.

Where did the nylon-eating ability come from? Carboxylesterases are enzymes with broad substrate specificities; they can carry out a variety of reactions. Their binding pocket is large and can accommodate a lot of different substrates. They are “promiscuous” enzymes, in other words. Furthermore, the carboxylesterase reaction hydrolyzes a chemical bond similar to the one hydrolyzed by nylonase. Tests revealed that both the EII and EII′ enzymes have carboxylesterase and nylonase activity. They can hydrolyze both substrates. In fact it is possible both had carboxylesterase activity anda low level of nylonase activity from the beginning, even before the appearance of nylon.

nylB′ may be the original gene from which nylB came. Apparently there was a gene duplication at some point in the past. The two genes appear to have acquired mutations since then — they differ by 47 amino acids out of 392. The time of that duplication is unknown, but not recent, because it takes time to accumulate that many mutations. However, at least some of those mutations must confer a high level of nylonase activity on EII, the enzyme made by nylB. The enzyme EII’ made by nylB’ has only a low ability to degrade nylon, while EII degrades nylon 1000 fold better. So one or more of those 47 amino acid differences must be the cause of the high level of nylonase activity in EII. Through careful work, the Japanese workers Kato et al. identified which amino acid changes were responsible for the increased nylonase activity. Just two step-wise mutations present in EII, when introduced into EII’, could convert the weak enzyme EII’ to full nylonase activity.

From Kato et al. (1991):

Our studies demonstrated that among the 47 amino acids altered between the EII and EII’ proteins, a single amino acid substitution at position 181 was essential for the activity of 6-aminohexanoate-dimer hydrolase [nylonase] and substitution at position 266 enhanced the effect.

So. This is not the story of a highly improbable frame-shift producing a new functional enzyme. This is the story of a pre-existing enzyme with a low level of promiscuous nylonase activity, which improved its activity toward nylon by first one, then another selectable mutation. In other words this is a completely plausible case of gene duplication, mutation, and selection operating on a pre-existing enzyme to improve a pre-existing low-level activity, exactly the kind of event that Meyer and Axe specifically acknowledge as a possibility, given the time and probabilistic resources available. Indeed, the origin of nylonase actually provides a nice example of the optimization of a pre-existing fold’s function, not the innovation or creation of a novel fold.

As the scientists who carried out the structural determination for nylonase themselves note:

Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the beta-lactamase fold resulted in the evolution of the nylon oligomer hydrolase. [Emphasis added.]

Let’s put to bed the fable that the nylon oligomer hydrolase EII, colloquially known as nylonase, arose by a frame-shift mutation, leading to the creation of a new functional protein fold. There is absolutely no need to postulate such a highly improbable event, and no justification for making this extravagant claim. Instead, there is a much more parsimonious explanation — that nylonase arose by a gene duplication event some time in the past, followed by a series of two mutations occurring after the introduction of nylon into the environment, which increased the nylon oligomer hydrolase activity of the nylB gene product to current levels. Could this series of events happen in forty years? Most certainly. Probably in much less time. In fact, it has been reported to happen in the lab under the right selective conditions. And most definitely, the evolution of nylonase does not call for the creation of a novel protein fold, nor did one arise. EII’s fold is part of the carboxylesterase fold family. Carboxylesterases serve many functions and have been around much longer than forty years.

Douglas Axe and Stephen Meyer readily admit that this kind of evolutionary adaptation happens easily. A protein that already has a low level of activity for a particular substrate can be mutated to favor that side reaction over its original one, often in just a few steps. There are many cases of this in the literature. What Axe and Meyer do claim is that generating an entirely new protein fold via mutation and selection is implausible in the extreme. Nothing in the nylonase story that Dennis Venema tells shows otherwise.

Tomorrow: “The Nylonase Story: How Unusual Is That?”

Photo: Nylon parachute, by Lance Corporal Brian D. Jones, U.S. Marine Corps [Public domain], via Wikimedia Commons.

Elegant.jpg

When certain biologists discuss the early stages of life there is a tendency to think too vaguely. They see a biological wonder before them and they tell a story about how it might have come to be. They may even draw a picture to explain what they mean. Indeed, the story seems plausible enough, until you zoom in to look at the details. I don't mean to demean the intelligence of these biologists. It's just that it appears they haven't considered things as completely as they should. Like a cartoon drawing, the basic idea is portrayed, but there is nothing but blank space where the profound detail of biological processes should be.

Let me give an example. This week Discovery Institute released a pair of videos ("How to Build a Worm" and "Switched on Worms") featuring Fellow and philosopher of biology Paul Nelson and a lowly nematode called C. elegans. Its development is precise and intricately patterned, like a Bach fugue that splits and weaves many voices into one. The final cadence is the newly hatched larva. Not coincidentally, the videos use music by Bach throughout.

C. elegans's development is a wonder to behold, well worth sharing with the world. But there is more: the development of these lowly worms exposes a major problem with the Darwinian account of evolution.

The development of C. elegans is an end-directed process. As the videos reveal, each embryo follows a precisely choreographed developmental road map in order to get to the final goal -- the reproductive adult. None of the intermediate steps on the way to the adult will do. Each step is necessary but not sufficient by itself. Turn aside from this developmental pathway and the result is likely to be a damaged worm or a dead one. Skip some steps and the same is true. How did this process come about? We would say this goal-directedness is evidence for a designer who had the final end in mind, and arranged the proper developmental steps appropriately.

Evolutionary biologists disagree. They say this exquisitely refined developmental pathway evolved gradually, a little at a time. First there was a cell, then a eukaryotic cell, one with a nucleus, organelles, and a cytoskeleton. Then along came multicellularity -- cells living together to make an organism, with some cells set aside to make the next generation, and others free to specialize. As time went on, new digestive, muscle, nerve, and sensory cells evolved and were successfully coordinated into functioning whole organisms.

These organisms then adopted increasingly complex shapes and lifestyles (including the lifestyle of the nematode worm), and developed spicules, chitin, fur or feathers, the ability to creep or to fly, to see and to sing. All these things happened because of random genetic change and natural selection. No road map, no design was needed. How do we know this happened? Because we can find a series of animals that resemble each step in the story -- Volvox, the sea urchin embryo, the planula of jellyfish, and flatworms like planaria. We find fossils that tell the story as more hard-bodied animals came along. The genes of these animals also speak of a common history (though the relationships are disputed for the deep histories). Finally, the series progresses logically, and purely by naturalistic means.

I should add that no one thinks Volvox, sea urchins, or flatworms (etc.) actually are ancestral to C. elegans or anything else. Rather these forms represent hypothetical "intermediates" that are discussed in a general way in textbooks (this may have happened, could have been, probably was...). Actual intermediates from unicellular eukaryotes to C. elegans are nowhere to be found, though. They are long gone, if they ever existed, so no one knows what they would have looked like. Despite this, names derived from these hypotheticals are assigned to clades, all based on genetic comparisons between extant species.

There's a problem, though. The story leaves out too much. To make this clear I have drawn a cartoon version (there are other versions, of course, because nobody really knows how it happened).

A cell. That step's a doozy (top left).

Cells stick together (like sponges?).

Cells form a hollow sphere with some set aside as reproductive cells, others for swimming and taking in food (like Volvox?).

Gastrulation. Cells move inward to form distinct layers and begin to differentiate one from another (like the sea urchin embryo?).

Another big step, a planula larva with bilateral symmetry, epithelium (a sheet of cells that encloses the body), a mouth, and perhaps a gut (like the planula of jellyfish? Not shown).

Over evolutionary time some planulae develop into something with a gut, epithelial, neural, muscular, and sensory cells (shown here as something like a planarium -- a small flatworm with eyespots, nerves, epithelia, muscle, and a one-way gut). Other planulae become the animals we call jellyfish or comb jellies. Then comes the evolution of all other animal forms as documented by the fossil record. Two products are shown: C. elegans and, by way of an early chordate with a notochord and gill slits, us.

This cartoon of the evolutionary story is stripped down to its bare essentials. Does it seem oversimplified? It is. Besides the fact that no one knows what happened, in biology nothing is so simple. Imagining a series of animals in such a story does not take into account or explain what is necessary for transitions within the series to take place. Similarity, or a progression in forms, does not mean there is an undirected pathway between those things. The details of how the transitions took place must be supplied, and their reasonableness assessed.

New genes and proteins must be invented. The cytoskeleton, Hox genes, desmosomes, cell adhesion molecules, growth factors, microtubules, microfilaments, neurotransmitters, whatever it takes to get cells to stick together, form different shapes, specialize, and communicate must all come from somewhere. Regulatory proteins and RNAs must be made to control the expression in time and space of these new proteins so that they all work together with existing pathways.

In fact, in order for development to proceed in any organism, a whole cascade of coordinated genetic and biochemical events is necessary so that cells divide, change shape, migrate, and finally differentiate into many cell types, all in the right sequence at the right time and place. These cascades and the resulting cell divisions, shape changes, etc., are mutually interdependent. Interrupting one disrupts the others. In C. elegans changing one cell division can have significant effects on the fates of downstream cells, and the earlier the change, the more downstream effects there will be. Coordinated, finely tuned cascades of events that lead to functional organisms cannot be produced by chance and natural selection alone.

If all these processes happen in the right way -- the road map is followed -- the cells of C. elegans successfully divide to make a worm that swims, eats, and reproduces, helping to turn compost into soil. If other creatures follow their own roadmaps, chicks hatch and caterpillars turn into butterflies, kittens are born, and babies sleep in their mother's arms.

According to evolutionary theory there is no roadmap, no design for how we got here. But after taking a closer look and filling in the details, it becomes apparent that there is a deep, coordinated, planned order in the world, as evidenced by C. elegans's development or the development of any creature -- an order with beauty and purpose, like a Bach fugue.

There's a problem, though. The story leaves out too much. To make this clear I have drawn a cartoon version (there are other versions, of course, because nobody really knows how it happened).

A cell. That step's a doozy (top left).

Cells stick together (like sponges?).

Cells form a hollow sphere with some set aside as reproductive cells, others for swimming and taking in food (like Volvox?).

Gastrulation. Cells move inward to form distinct layers and begin to differentiate one from another (like the sea urchin embryo?).

Another big step, a planula larva with bilateral symmetry, epithelium (a sheet of cells that encloses the body), a mouth, and perhaps a gut (like the planula of jellyfish? Not shown).

Over evolutionary time some planulae develop into something with a gut, epithelial, neural, muscular, and sensory cells (shown here as something like a planarium -- a small flatworm with eyespots, nerves, epithelia, muscle, and a one-way gut). Other planulae become the animals we call jellyfish or comb jellies. Then comes the evolution of all other animal forms as documented by the fossil record. Two products are shown: C. elegans and, by way of an early chordate with a notochord and gill slits, us.

This cartoon of the evolutionary story is stripped down to its bare essentials. Does it seem oversimplified? It is. Besides the fact that no one knows what happened, in biology nothing is so simple. Imagining a series of animals in such a story does not take into account or explain what is necessary for transitions within the series to take place. Similarity, or a progression in forms, does not mean there is an undirected pathway between those things. The details of how the transitions took place must be supplied, and their reasonableness assessed.

New genes and proteins must be invented. The cytoskeleton, Hox genes, desmosomes, cell adhesion molecules, growth factors, microtubules, microfilaments, neurotransmitters, whatever it takes to get cells to stick together, form different shapes, specialize, and communicate must all come from somewhere. Regulatory proteins and RNAs must be made to control the expression in time and space of these new proteins so that they all work together with existing pathways.

In fact, in order for development to proceed in any organism, a whole cascade of coordinated genetic and biochemical events is necessary so that cells divide, change shape, migrate, and finally differentiate into many cell types, all in the right sequence at the right time and place. These cascades and the resulting cell divisions, shape changes, etc., are mutually interdependent. Interrupting one disrupts the others. In C. elegans changing one cell division can have significant effects on the fates of downstream cells, and the earlier the change, the more downstream effects there will be. Coordinated, finely tuned cascades of events that lead to functional organisms cannot be produced by chance and natural selection alone.

If all these processes happen in the right way -- the road map is followed -- the cells of C. elegans successfully divide to make a worm that swims, eats, and reproduces, helping to turn compost into soil. If other creatures follow their own roadmaps, chicks hatch and caterpillars turn into butterflies, kittens are born, and babies sleep in their mother's arms.

Studying the design of life

We knew organization had to be a feature of DNA in the nucleus, but now we have a fine scale picture of exactly how organized it is.