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Chemical Biology

Evolving Enzymes: Let ‘Em Rip

Evolutionary and genetic processes fascinate many organic chemists, and with good reason. They’ve provided us with the greatest set of chemical catalysts we know of: enzymes, which are a working example of molecular-level nanotechnology, right in front of us. A billion years of random tinkering have accomplished a great deal, but (being human) we look at the results and wonder if we couldn’t do things a bit differently, with other aims in mind than “survive or die”.
This has been a big field over the years, and it’s getting bigger all the time. There are companies out there that will try to evolve enzymes for you (here’s one of the most famous examples), and many academic labs have tried their hands at it as well. The two main routes are random mutations and structure-based directed changes – and at this point, I think it’s safe to say that any successful directed-enzyme project has to take advantage of both. There can be just too many possible changes to let random mutations do all the work for you (20 to the Xth power gets out of hand pretty quickly, and that’s just the natural amino acids), and we’re usually not smart enough to step in and purposefully tweak things for the better every time.
Here’s a new paper that illustrates why the field is so interesting, and so tricky. The team (a collaboration between the University of Washington and the ETH in Zürich) has been trying to design a better retro-aldolase enzyme, with earlier results reported here. That was already quite an advance (15,000x rate enhancement over background), but that’s still nowhere near natural enzymes of this class. So they took that species as a starting point and did more random mutations around the active site, with rounds of screening in between, which is how we mere humans have to exert selection pressure. This gave a new variant with another lysine in the active site, which some aldolases have already. Further mutational rounds (error-prone PCR and DNA shuffling) and screening let to a further variant that was over 4000x faster than the original enzyme.
But when the team obtained X-ray structures of this enzyme in complex with an inhibitor, they got a surprise. The active site, which had already changed around quite a bit with the addition of that extra lysine, was now a completely different place. A new substrate-binding pocket had formed, and the new lysine was now the catalytic residue all by itself. The paper proposes that the mechanistic competition between the possible active-site residues was a key factor, and they theorize that many natural enzymes may have evolved through similar paths. But given this, there are other questions:

The dramatic changes observed during RA95 evolution naturally prompt the question of whether generation of a highly active retro-aldolase required a computational design step. Whereas productive evolutionary trajectories might have been initiated from random libraries, recent experiments with the same scaffold dem- onstrate that chemical instruction conferred by computation greatly increases the probability of identifying catalysts. Although the programmed mechanisms of other computationally designed enzymes have been generally reinforced and refined by directed evolution, the molecular acrobatics observed with RA95 attest to the functional leaps that unanticipated, innovative mutations—here, replacement of Thr83 by lysine—can initiate.

So they’re not ready to turn off the software just yet. But you have to wonder – if there were some way to run the random-mutation process more quickly, and reduce the time and effort of the mutation/screening/selection loop, computational design might well end up playing a much smaller role. (See here for more thoughts on this). Enzymes are capable of things that we would never think of ourselves, and we should always give them the chance to surprise us when we can.

14 comments on “Evolving Enzymes: Let ‘Em Rip”

  1. pgwu says:

    They went on the road least traveled nowadays and had a great discovery.

  2. eugene says:

    On to the next challenge. Now they can try to evolve DNA that is made up of arsenic and doesn’t collapse after one second. I know a postdoc with NASA funding that might be interested in the project.
    Actually, more seriously, I’ve been wondering for a long time whether it’s possible to evolve enzymes (and bacteria by extension) to use second row transition metals in order to do some novel reactions that you can’t see in nature for now. I think that the reason nature didn’t do this is purely due to abundance. First row metals are abundant, but you would have trouble surviving as a Pd bacteria, even if the enzymes simplified a lot of the synthetic pathways. A pool rich in Pd and a billion years might result in some interesting lifeforms, but these conditions have never existed on earth and Pd is probably a poison for most living things and introducing it now is an insurmountable selection pressure these days for something living.

  3. Aleksandra says:

    eugene, perhaps it is a silly question, but what do you think this reactions (with the use of second row transition metals) would look like? I mean, do you think about something specific?

  4. Anonymous says:

    eugene, there is a cadmium-containing carbonic anhydrase – but it doesn’t do “novel reactions that you can’t see in nature”:
    Nature. 2008 Mar 6;452(7183):56-61. doi: 10.1038/nature06636.
    Structure and metal exchange in the cadmium carbonic anhydrase of marine diatoms.
    Xu Y, Feng L, Jeffrey PD, Shi Y, Morel FM.
    Carbonic anhydrase, a zinc enzyme found in organisms from all kingdoms, catalyses the reversible hydration of carbon dioxide and is used for inorganic carbon acquisition by phytoplankton. In the oceans, where zinc is nearly depleted, diatoms use cadmium as a catalytic metal atom in cadmium carbonic anhydrase (CDCA). Here we report the crystal structures of CDCA in four distinct forms: cadmium-bound, zinc-bound, metal-free and acetate-bound. Despite lack of sequence homology, CDCA is a structural mimic of a functional beta-carbonic anhydrase dimer, with striking similarity in the spatial organization of the active site residues. CDCA readily exchanges cadmium and zinc at its active site–an apparently unique adaptation to oceanic life that is explained by a stable opening of the metal coordinating site in the absence of metal. Given the central role of diatoms in exporting carbon to the deep sea, their use of cadmium in an enzyme critical for carbon acquisition establishes a remarkable link between the global cycles of cadmium and carbon.

  5. Anonymous says:

    Here’s another wacky study:
    Nature. 2010 Aug 5;466(7307):779-82. doi: 10.1038/nature09265. Epub 2010 Jul 18.
    Microbial metalloproteomes are largely uncharacterized.
    Cvetkovic A, Menon AL, Thorgersen MP, Scott JW, Poole FL 2nd, Jenney FE Jr, Lancaster WA, Praissman JL, Shanmukh S, Vaccaro BJ, Trauger SA, Kalisiak E, Apon JV, Siuzdak G, Yannone SM, Tainer JA, Adams MW.
    Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.

  6. eugene says:

    “eugene, perhaps it is a silly question, but what do you think this reactions (with the use of second row transition metals) would look like? I mean, do you think about something specific?”
    I was thinking of weird stuff, like oxidative methane coupling and later alkane metathesis for a methane-seep based bacteria to store energy to use for later in the form of liquid fuel. Maybe even CH activation of specific substrates and subsequent coupling to a halide modified natural product. Or even something that can do olefin metathesis (Ru based) for a very specific purpose.
    Anonymous, thanks for the papers!

  7. tt says:

    Eugene: Here’s a paper from the Rovis group that engineered Rh(III) into an enzyme active site to do S-H activation.
    Francis Arnold (Caltech) has also done extensive work to alter BM3 oxidase to acept substrate larger than methane.

  8. tt says:

    Oops…I meant C-H activation

  9. eugene says:

    Thanks tt! I completely forgot about the Rovis paper. I think a friend of mine sent it to, because I kept talking about just this type of thing, me when it came out, but I don’t remember how I came across it back in the day.
    That’s exactly what I was thinking of; it’s an excellent paper. But like a lot of first-principle Science articles where I know what’s going on (like the Goldman alkane metathesis, Milstein water splitting, etc…) it seems like it’s a bit of a low-hanging fruit kind of thing. A real advance is still far away and it’s just proof of principle. Here the enzyme has a binding pocket on the outside that is set up to take pretty much any metal and you expect it to effect ee just like any other chiral ligand.
    I don’t want it to seem like I’m complaining, because it’s a good paper, but still… I feel like the full potential of evolving and engineering metallo-enzymes to take advantage of transformations that we don’t see in nature, or even have a living organism that takes advantage of all that, is still an area in which a lot of novel research can be done. There are just a lot of tools still missing.

  10. Kurt says:

    I agree with Eugene. The potential of artificial metalloenzymes is far from being fully realized or appreciated. I would disagree with the comment that the enzyme is simply doing what any other chiral ligand is capable of. In the Rovis/Ward paper, an acetate is also introduced into the active site to provide enhanced activity of the complex. Given the modest er reported in the paper and the relatively small substrate scope, the enhanced activity seems to be the most interesting part of their paper. In terms of having these systems applied by living organisms, I see there being a potential issue of cellular compounds coordinating to free coordination sites on the metal. Of course you could evolving solutions to this.

  11. tt says:

    I agree with you completely Eugene. It’s yet to be shown whether we can really engineer a transition metal binding enzyme with new reactivity patterns. Given the current focus on base metal catalysis, I would really like to see someone develop a C-H cross coupling type reaction with just iron. From a cost and perhaps selectivity perspective, this would be a real advance. One of the current limitations of most biocatalysis is that they don’t really take two big, greasy reactants and couple them together. Nature tends more towards long linear synthetic sequence (think polyketides).

  12. Kent Kemmish says:

    If you like it, share it.
    Evolution is smarter than any of us. This is why I’m obsessed with sequence space. This is why it’s more important than outer space. I’m tired of the refrain I’ve heard more than once that directed evolution must go hand-in-hand with careful scaffolding and computational strategies and that there’s really no way to get from non-active to active without a lot of clever guidance. Bollocks! Unleash the genius idiocy of evolution and put your ego aside.
    The path to industrialized molecular engineering is right there in front of you. But… “It takes a great general to see the obvious”.
    I’m blessed to know so many brilliant people, but to me the very brightest of them all share a certain intellectual honesty- a type of humbleness- that somehow correlates to being able to see the obvious in that sense.
    I wish I could pour my heart and my vision out to all of them, and get the help I need to launch us as deep into sequence space as we should have already gone. My vision is like the night sky of Sequence Space- so many little twinkling monsters of wonder, and I feel that with the vast majority of my colleagues don’t even realize that they’re just there to be visited by us almost as a matter of willing it to be so.
    We (the apostles of the Church of Sequence Space, and Keltar) need a lab to work in where we can prove some of our ideas about Sequence Space exploration. The lab should have access to next-gen sequencing and synthesis. If we can do both more or less continuously for six months to a year, we will reach the stars. Both sequencing and synthesis are already good enough to enable this.
    I believe fanatically that we can accomplish all of the following in less than a year with access to such infrastructure. The avant-garde of the directed evolution and molecular engineering literature supports this assertion, but so few people are crazy enough to grok it all at once…
    The galaxy of stars that can be reached in less than one year contains:
    1) diamond-building enzymes
    2) huge libraries of diverse alkyating enzymes, including enzymes that build long alkane chains from CO2, water, and sunlight (i.e., gasoline growing enzymes)
    3) fullerene building enzymes (how do we build a space elevator with carbon nanotubes? We grow it concentrically. )
    4) huge libraries of biomedical remediation enzymes for glycoSENS, lysoSENS, and amyloSENS
    5) sub-femtomolar affinity aptamers
    6) genomic editing enzymes
    7) ice-blocking reagents for improved vitrification
    And these are just the ones I’m confident about.
    I need: 1) an Ion Proton, though an Ion Torrent will probably do,- an Illumina rig would also work, 2) a set-up for micro-mirror directed oligo synthesis and release (100-mers will suffice for everything), 3) a modest budget for reagents (~$5K/month), 4) a few fanatic knowledgeable folks like Carl Crott to help out- just two or three Carls would be all I would need at most, and 5) a stipend of just a few grand a month for each of us in order to survive and not be on unemployment.
    If I can find a lab that already has the sequencing and synthesis infrastructure, then I’m looking for support on the order of only $100K for getting to the stars of sequence space in less than a year. If I have to set up a lab and buy sequencing and synthesis, then I’m looking at about $500K for the year. Bear in mind that just one of those seven stellar deliverables I listed would be worth many billions of dollars.
    Who can offer advice, assistance, support? Is there anyone out there who could or would help financially and would be openminded, given that I don’t expect a scrawl on facebook to result in funding, but rather that I’d want you to engage with me in a dialogue?
    Is there anyone who can recommend a lab where I might find a home again?

  13. srp says:

    Tried Kickstarter or Indiegogo?

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