I can’t find my way through the thicket of threads to say what I want to say so I will say it here. You seem to have found quite a bit of the relevant stuff on your own, i.e. the Jurgens paper. If you read that paper carefully you will see that they point out that HisA and TrpF carry out the same reaction, but with different R groups. They have the same basic chemistry, As I have said before, enzymes that have the same basic chemistry can frequently be shifted toward the other’s function. This is not a surprise. Likewise, if you have an enzyme that has two catalytic activities, subfunctionization can occur. This is not a surprise. If duplicating a gene is beneficial, that too, will occur. Not a surprise.
So to let you know how not a surprise it all was, I was gathering up references. You have found some already. I will provide others. But before I do, let me lay out the hierarchy of difficulty of evolution of an enzyme to a new function, from easiest to hardest. There must be a selectable level of starting function or function within one or two steps and
- Overlapping function, shared chemistry: promiscuous enzyme or
- Shared chemistry (types of bond broken and formed, mechanisms shared.)
- Substrates in common, but no shared reaction types are unlikely to work.
- No shared function, chemistry or substrates won’t work.
The kinds of successful conversions you see in the literature are almost always 1 or 2. If you don’t believe me, look carefully. Read the methods and check for baseline activity before conversion like Rumraket did. I would have pointed it out if he hadn’t.
BioF and Kbl were type #4. That was our purpose, to see if it was possible to convert an enzyme with no chemistry or substrate preference in common to another enzyme’s function. As trpF and HisA have shown, sequence identity doesn’t matter. Apparently shared chemistry does. That and the kind of fold the enzyme has, a beta barrel, with the active site located external to the barrel, on unstructured loops, accessible and easy to modify. BioF and Kbl have a much more complicated reaction and active site.
Nasvall et al was a fine study. It used a great deal of previously gathered knowledge about these genes and their regulation to make sure they could make the double function enzyme (they designed and carried out the selection–they created it, even though they did not pick the mutations), and then get it to express first in one condition and then the other without interference. Could that his regulatory mutant have happened on its own? Sure. But the sequence, starting with the set up in a cassette to guarantee constitutive expression, and the selection to split trpC and trpF and then get the double function enzyme, and then to improve it, and then to allow expression in the presence of histidine, and then the extended culture without interference from competing wildtype cells… It’s a nice proof of concept experiment but it won’t happen on its own. How many steps? 6? This is what I was upset about. Just because you can get something to happen in a lab does not mean it will happen for real. How many of those steps would be beneficial in a mud puddle or intestine? We know some bacteria have dual function genes, so it can happen, but not by this elaborate path.
Maybe this is my complaint in a nutshell. You all seem to assume I think this kind of thing won’t happen, that gene duplication and recruitment never works, or exaptation. It does, but under limits.
And that leads to the other ridiculous thing about the use of Nasvall in the first place. Behe himself doesn’t disagree with Nasvall et al. He said so in his review. Those who tried to say it disproved his work misunderstand his work
I never said or implied that a conversion like Nasvall et al described could not work. That would be ridiculous. These were papers I looked at when we were considering what system to use.
From an early draft of the bioF paper:
While numerous experimental studies have aimed to demonstrate the feasibility of reconfiguration, several complicating factors tend to be overlooked when results are interpreted. First, there is surprisingly little discussion of how evolutionary feasibility falls off as the number of required mutations increases. Schmidt and coworkers pay more attention to this than most, observing that “if natural divergent evolution of mechanistic diversity is to be accomplished, only a limited number of substitutions can be required for selective advantage; otherwise, the probability of accumulating the necessary mutations would be too low” [12]. But while they claim that most experimental demonstrations require too many nucleotide substitutions, they offer no way to draw a line between what is feasible and what is not.
Another point, which they also raise, is that many demonstrations of enzyme reconfiguration merely modify substrate specificities without changing the mechanism of reaction. In most cases the modifications simply shift the relative preference for existing substrates (see for example [13–17 ]), but even when mutations cause enzymes to recognize genuinely new substrates [18–19 ], this restricted category of diversification leaves the most important kind of metabolic innovation—new pathways built on new chemistries—unexplained.
Modify this to mention recent papers describing very low limits for feasible transitions, including your manuscript in review.
Ref 19 belongs here: Jurgens et al 2000 and add Leopoldseder et al 2004
Do you mean same chemistry, new substrates, or do you mean new chemistry here? I have given refs for new chemisrty in the next comment. For a case where the chemistry is similar but the substrate different, and near wild-type activity was achieved, see Claren et al 2009 in HisA Trp F folder( Establishing wild-type levels…)
New chemistry: Cite Toscano et al for a review, plus 18, Xiang et al 1999, Schmidt et al 2003, Ma and Penning 1999, Vick et al 2007 (this paper describes optimization of OSBS with four mutations). Delete 19 here.
And additional reference on trpF and HisA
Enzymes are known to be amazingly specific and efficient catalysts. However, many enzymes also have so-called promiscuous functions, i.e., they are able to catalyze other reactions than their main one. The promiscuous activities are often low, serendipitous, and under neutral selection but if conditions arise that make them beneficial, they can play an important role in the evolution of new enzymes. In this thesis, I present three studies where we have characterized different enzyme families by structural and biochemical methods. The studies demonstrate the occurrence of enzyme promiscuity and its potential role in evolution and organismal adaptation. In the first study, I describe the characterization of wild type and mutant HisA enzymes from Salmonella enterica. In the first part of this study, we could clarify the mechanistic cycle of HisA by solving crystal structures that showed different conformations of wild type HisA in complex with its labile substrate ProFAR (N´-[(5´-phosphoribosyl)formimino]-5- aminoimidazole-4-carboxamide ribonucleotide). In the second part of this study, structures of mutant enzymes from a real-time evolution study provided us with an atomic-level description of how HisA had evolved a new function. The HisA mutants had acquired TrpF activity, either in addition to (bifunctional generalists) or instead of (TrpF specialists) their HisA activity.
https://onlinelibrary.wiley.com/doi/full/10.1038/sj.embor.embor771
We report the occurrence of an isomerase with a putative (βα)8‐barrel structure involved in both histidine and trypto‐phan biosynthesis in Streptomyces coelicolor A3(2) and Mycobacterium tuberculosis HR37Rv. Deletion of a hisA homologue (SCO2050) putatively encoding N ′‐[(5′‐phosphoribosyl)‐formimino]‐5 amino‐imidazole‐4‐carboxamide ribonucleotide isomerase from the chromosome of S. coelicolor A3(2) generated a double auxotrophic mutant for histidine and tryptophan. The bifunctional gene SCO2050 and its orthologue Rv1603 from M. tuberculosis complemented both hisA and trpF mutants of Escherichia coli . Expression of the E. coli trpF gene in the S. coelicolor mutant only complemented the tryptophan auxo‐trophy, and the hisA gene only complemented the histidine auxotrophy. The discovery of this enzyme, which has a broad‐substrate specificity, has implications for the evolution of metabolic pathways and may prove to be important for understanding the evolution of the (βα)8‐barrels.