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Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: Twitter: Dereklowe

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August 16, 2011

Screening Quickly Through the Mutants

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Posted by Derek

Here's another paper at the intersection of biology and chemistry: a way to check the activity of a huge number of mutated esterase enzymes, all at the same time.

Protein engineering is a hot field, as well it should be, since enzymes do things in ways that we lowly organic chemists can only envy. Instead of crudely bashing and beating on the molecules out in solution, an enzyme grabs each of them, one at a time, and breaks just the bond it wants to, in the direction it wants to do it, and then does it again and again. If you're looking for molecular-scale nanotechnology, there it is, and it's been right in front of us the whole time.

Problem is, enzymes get that way through billions of years of evolution and selection, and those selection pressures don't necessarily have anything to do with the industrial reactions we're thinking of these days. And since we don't have a billion years to wait, we have to speed things up. Thus the work at places like Codexis on engineered mutant enzymes, and thus a number of very interesting takes on directed evolution. (Well, interesting to me, at any rate - I have a pronounced weakness for this sort of thing).

This latest paper, from the University of Greifswald in Germany, builds on the work of Manfred Reetz at the Max-Planck Institute, who's been very influential in the field. Specifically, it follows up on the idea in this paper from his group and this one from the Quax group at Groningen in the Netherlands. That technique involved selecting for specificity in esterase enzymes by giving organisms a choice of two substrates: if they hydrolyze the right chiral starting material, the cleaved ester furnishes them with a nutrient. If they hydrolyze the wrong one, though, they produce a poison. Rather direct, but with bacteria there's no other way to get their attention - survival's really all they care much about.

And that technique worked, but it was a bit laborious. The largest number of different variations tested was about 2500, which seems like a lot until you do the math on protein mutations. It gets out of control very, very quickly when you have twenty variations per amino acid residue. Naturally, some of the residues shouldn't ever be touched, while others will have only minimal effects, and others are the hot spots you should be concentrating on. But which ones are which? And since you absolutely can't assume that they're all acting independently of each other, you have your work cut out for you. (Navigation through this thicket is what Codexis is selling, actually).

This latest paper adds flow cytometry, cell sorting, to the mix. Using dye systems and one of these machines to distinguish viable bacteria from dead or dying ones lets you take a culture and pull out only the survivors. When the authors expressed different esterases (with known preferences for the two substrates) in E. coli, they got the expected results - the ones with an enzyme that could cleave the nutrient-giving substrate grew, while the ones that unveiled the poison (2,3-dibromopropanol) halted in their tracks.

They then took another esterase with very modest selectivity and created a library of mutant variations - about ten million mutant variations - and expressed the whole shebang in a single liquid colony of E. coli. This was then exposed to the mixture of substrates, and anything that grew was pulled out by the cell sorter and plated out on agar (also containing the selection mixture of substrates). They got 28 clones to grow in the end, and characterized three of these more fully as purified enzymes. Of those, two of them were, in fact, much more selective than the starting enzyme (giving E values, enantiomeric ratios, of 80 to 100 as opposed to 3). Another, interestingly, was not selective at all.

And when you look at the sequences and the mutations that were picked out, you can see how tricky a business this is. One of the two selective enzymes had its valine-121 residues mutated to isoleucine (V128I) its phenylalanine-198 residue mutated to cysteine (F198C). The other was broadly similar, with one added mutation: that valine-121 was changed in this case to serine (V121S), the F198 was mutated to glycine (F198G), and also valine-225 was changed to alanine (V225A). Now, some of those aren't very big mutations (V to I, V to A), but what's even more interesting is the sequence of the unselective clone that they characterized: that one had V121I, F198G, V225A. So it had a mix of the exact mutations found in the two selective enzymes, but was itself a dud.

I'm glad to see that this worked, although you have to wonder how efficiently it moves in on a target when you get two decent hits out of ten million starting mutations. (The relative ease of screening goes a long way towards making up for that). But what I'd like to see is a mix of this technique with the one that I wrote about a few weeks ago, where a bacterium was evolved to use a chlorinated DNA base. That one used a particularly slick directed-evolution device, which would be quite interesting to apply to this food-versus-poison idea. You'd have to do some fine-tuning, especially at first, since the liberated poisonous substrate would be killing off the just and the unjust alike (which is the same problem that this current paper faced). But it seems like there should be a way to run things so that you're not just screening a big library of random mutations in the enzyme, but actually pushing the enzyme to evolve in the direction you want. Thoughts?

Comments (12) + TrackBacks (0) | Category: Chemical Biology


1. Tt on August 16, 2011 11:27 AM writes...

Very clever indeed! The could be a really quick and easy way to engineer activity and identify "hot spots". It could then be followed with more focused mutation libraries. The only downside is that this idea really only works with hydrolyses. It may be trickier to couple a separate nutrient cycle to reductases, oxidases, or transferases.

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2. Alex Besogonov on August 16, 2011 1:32 PM writes...

I'm curious, are there any studies to design an enzyme de-novo from the first principles? Even a simple 'Hello, world!'-style enzyme?

That would be awesome.

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3. Shazz on August 16, 2011 5:50 PM writes...

I have a weakness for this field also.

Does turning to directed evolution mean that the golden age of reductionist rationalism is over? The trend of using streamlined trial and error seems to be emerging not just in protein science.

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4. leftscienceawhileago on August 16, 2011 11:45 PM writes...

That is a common desire amongst many people. The problem is, there isn't really such a "hello world" molecule.


Because any interesting molecular phenomenon ends up just being a many body problem, and often, many properties only manifest themselves when considering distributions over large populations of molecules in the context a bulk phase, which is difficult to model in general. In fact it is very difficult to make certain types of predictions even considering a simple molecule in isolation (look up the Schrodinger wave equation).

There is some basic reasoning and rules that you can "apply by eye" when it comes to thinking about proteins (or molecules in general) e.g. 'oily' residues want to be buried inside, shielded from the water environment while 'polar' residues want do the opposite.

This type of reasoning really just helps you test for consistency, i.e., a model you are working with plausible. But these rules of thumb aren't really 'constructive'.

Could we have designed GFP (a protein that almost magically folds into a shape that catalyzes an intramolecular reaction that allow it to fluoresce green) if we didn't find it in nature first? No.

Can we design an enzyme that binds a small molecule of choice selectively? Well, lot's of model studies on this, but there isn't a single example of something that really was designed from first principles and blew people's minds. Hellinga's work aspired to this, but unfortunately it was all bunk.

Can we design a protein that can use ATP to fuel it's own replication from fragments? Nope.

We have all sorts of studies that have make strides towards goals like these, but not general methods that make it as simple as pressing a button. The reason why is precisely what I said above, you are trying to solve a many body problem and that is difficult to do. There have been some neat strides in predicting structures, even those that are not homologous to natural proteins; but turning those structures into ones that do something cool isn't easy.

The 'simple' way proteins are coded in strings make it tempting to think of protein sequences as little programs that do our bidding; sadly, it is not that simple, the sequence really just encodes that initial state of system that we are just getting our minds around methods to simulate accurately.

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5. Morten G on August 17, 2011 3:23 AM writes...

@2. Alex
Yeah a while back there were some people who made an alpha helical bundle from first principles. It did not blow my mind.

With regard to the problem of toxins killing bacteria that didn't produce the toxin: if they used a reactive toxin it wouldn't release from dead bacteria and kill more.

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6. simpl on August 17, 2011 8:06 AM writes...

Surely, also, training microorganisms should be the elegant way to make derivatives of the recently discussed macrolides - far better than opening and closing rings chemically.

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7. simpl to schazz on August 17, 2011 8:11 AM writes...

Streamlined trial and error is called numeric methods in engineering. It is a way to solve problems when models don't work.
Newton was quite happy to use it alongside calculus, for instance.

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8. David on August 17, 2011 9:51 AM writes...

This work is a good example of the process of directed evolution in action, and you provided an excellent summary of how the process works. Directed evolution is very powerful becuase it allows a catalyst (specifically an enzyme) to be tailored to desired reactions conditions, which as you point out, are not typically found in natural environments. High-throughput screening speeds up the process of directed evolution to the degree that it is now feasible to develop custom catalysts cost-effectively. This is why biocatalysis is expanding in scope, bringing renewable, non-toxic catalysts to a wider range of chemical processes.

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9. PharmaHeretic on August 17, 2011 11:53 AM writes...

I bet chemists are too chicken to pull of something like this.

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10. Curt F. on August 17, 2011 1:45 PM writes...

This paper is about as close to a first-principles *enzyme* design that I have seen.

1. The active site was designed de-novo using quantum mechanics calculations (and other calculations/methods too).

2. The catalyzed reaction is not known to biology.

3. What was not completely de-novo was finding a backbone to fold into the desired shape such that the active active site was formed. They relied on a database search to find good initial backbone structures. But it is close enough for me.

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11. Alex Besogonov on August 18, 2011 9:55 AM writes...

Curt F.:

Wow. Thanks! I'll definitely read this paper.

Permalink to Comment

12. Billyziege on August 18, 2011 11:43 AM writes...

Just a little number typo. Derek, you wrote:

"One of the two selective enzymes had its valine-121 residues mutated to isoleucine (V128I)"

Otherwise, thanks again for bringing interesting stuff to our attention.

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