Proteins are chains of amino acids, and each link in the chain can hold any one of the 20 amino acids that life relies on. If you were to pick each link at random, the number of possible proteins ends up reaching astronomical levels pretty fast.

So how does life ever end up evolving entirely new genes? One lab has been answering that question by making its own proteins from scratch.

Way back in 2016, the same lab figured out that new, random proteins can perform essential functions. And those new proteins were really new. They were generated by scientists who made amino acid sequences at random and then kept any that folded into the stable helical structures commonly found in proteins. These proteins were then screened to see if any could rescue E. coli that were missing a gene essential to survival.

Three proteins succeeded, which indicates that they compensated for the missing gene’s essential function. But they did not do so by acting as a catalyst (meaning they weren’t enzymes).

In a recent paper in Nature Chemical Biology, however, the lab is reporting that one newer protein has acted as a catalyst.

The E. coli used in these experiments lacked the ability to use the iron provided in their medium because of the deletion of a gene that normally provides this function. So the experiments were a test to see if a randomly generated protein would be able to catalyze reactions with iron. The three proteins that had passed this test in 2016, however, simply altered gene activity so that the iron became available through other pathways.

To generate the recent enzyme, the researchers took one of the proteins that already rescued the mutant E. coli and subjected it to random mutagenesis. This ultimately produced an iron-releasing enzyme. Just like the natural enzyme, this synthetic one has a chiral preference for its substrate, meaning that it can only work with one structural form of the molecule and not its mirror image.

But its similarities to the native enzyme end there. The amino acid sequence of this synthetic enzyme bears no relation to the bacterial enzyme it replaces. This made figuring out how it works very difficult. Usually this is done by comparing the protein in question to similar ones from other species: clearly not an option here. The researchers also tried to crystallize it, which would let them figure out its structure, but no deal.

So they started mutating amino acids one by one to see which mutations rendered the enzyme inactive. This told them that the original amino acid that had been replaced must be important. This method revealed five particular amino acids that comprise the likely active site. When software that predicts protein structures was given the protein’s amino acid sequence and told that these five had to be close together, it spit out one structure that seemed the most likely.

And just like the amino acid sequence, the structure looked so totally different from the native enzyme’s that the researchers think the enzyme must work through a completely new mechanism.

The scientists made this enzyme not using any kind of rational design or strategy; they were just tooling around with random amino acid sequences and having bacteria determine if they could do what they wanted. In a completely contrived case of convergent evolution, the researchers made a protein that does not share a sequence, structure, or even mechanism with the one evolution hit upon—yet it performs the same function. A thousand-fold slower than the natural one, but it might get better if given further time to evolve.

Nature Chemical Biology, 2018. DOI: 10.1038/NCHEMBIO.2550 (About DOIs).