@Mercer @Swamidass @Art @T_aquaticus @John_Harshman hn
I do not have a problem with this paper per se. My problem is you lot acting like this refutes Axe’s work .
Let me explain. This shouldn’t be hard, since you are all good, knowledgable scientists. The 5 clones they found were reported to have measured levels of activity against a known amount of substrate and phage, but the authors admit themselves this is not quantitative, because they do not know how many Abenz there are per phage, and it varies. No conclusion can be reached about the relative strengths of the reactions without some means of standardization.
The positive control, B-lactamase, was good, the best they could do, and the negative was understandable (empty phage), but it should have been what they started with (phage with unselected Abenz) in order to know how much of the hydrolysis was due to the inherent structure of the Abenz display and how much was due to selection of the Abenz. Whenever I did a library screen, the negative control was the plasmid plus vector starting point, not plasmid alone).
But my main problem is your claim that they have shown that Doug’s paper is wrong. Doug’s paper showed the rarity of a functional protein with a particular activity (B-lactam) and a particular structure ( TEM-1 B-lactam) (that’s what he and I mean by a functional fold BTW). Out all possible protein structures only 1 in 10^77 will have that structure and that enzymatic activity. It’s a way of answering the question, how many ways are there to make a protein that has that particular structure with that particular chemistry out of all possible proteins. It’s a question of some interest to protein scientists, and of necessity, to evolutionary biologists. Otherwise you wouldn’t care.
Now, this paper has shown that out of a library of 1 in 10^10 Abenz, when stabilized by binding to phage, you can obtain 5 clones with an active site capable of breaking down penicillin or analog of penicillin. Based on modeling, it is possible these five clones share in the kind of active site common to serine proteases (and beta lactams)- a triad of three particular amino acids held in the right orientation that causes hydrolysis of penicillin. But this is a proposed structure, based on a model, not a structural determination.
It turns out that breaking down penicillin is not hard. It hydrolyzes in water fairly rapidly. Anyone who works with it in the lab knows you need to take special precautions to be sure you have the same specific activity from experiment to experiment. They hint at that in this paper, saying something in passing about one of their fluorophores spontaneously hydrolyzing " In the fluorometric assay using Fluorocillin
TM, the penicillinase enzyme displays a Michaelis–Menten behavior with an initial slope of
19.7 min-1, whereas the negative controls, WT phage, as well as the spontaneous hydrolysis of FluorocillinTM, do not."
It would have been very nice to have all of that in a table. How can I tell that it displays Michaelis-Menten behavior if they don’t provide the data?
There’s another comparative problem. Things that work in the test tube often don’t in vivo. This is relevant because this paper describes an in vitro beta lactam; should they ever get a stable active form in vitro, it may do nothing in vivo. Just ask around, It’s a biotech nightmare come true. Doug’s measurements were in vivo.
HERE’s THE MAIN POINT. Suppose I grant they have a genuine active site displaying Michaelis-Mentin kinetics (which I don’t grant, all they provide is a slope), they still haven’t solved the problem of getting a 3D fold to stabilize the active site. The Ab enzyme is supported by a firm well established phage and is free to explore sequence space from that vantage point. They also were not constrained as to what way they could solve the problem. There are multiple ways to get a beta lactam.
This is significant. Doug’s experiment has always been about how hard it is to a a particular fold ( yes, I know that means structure, that’s what I mean!) with a particular function. He never claimed to have found the all structures that could act as a Beta lactam. He found how hard it is to get a TEM-1 beta-lactam enzyme.
If this group is able to create ABenzyme that is not phage-stabilized but is stabilized by its own 3D fold, and can measure its kinetics etc, that will be great. I am not dissing their work. I just don’t find it to be a refutation of Doug’s paper.
I am more than a bit disappointed in the way this paper has been presented here. I think some critical thinking was missing in the desire to score a victory. I have been accused of reading superficially, and it has been true. I have been guilty of reading just until I find the part that makes my point. Mea culpa. But think some of that has gone on here for some of you. I will not speculate as to motives.
But let me ask this. What would you make of this paper if it was about tetracycline?