By Oreo
Science correspondent and manuscript supervisor
Last week, Oreo interviewed the MBAB Lab team about their new chapter in Methods in Enzymology. The chapter brings together more than ten years of research, hard work, and exciting discoveries on the structure, function, and biotechnological applications of acetohydroxyacid synthase (AHAS).
There are days when Lili, Mario and Vivian sit in front of their computers and write for hours. I normally interpret this as a fairly clear sign that they have forgotten their priorities: me. But over the past few months, something was different. The same acronym kept appearing on their screens: AHAS. There were also names such as Arabidopsis thaliana, Saccharomyces cerevisiae, ThDP, FAD, penoxsulam, oxidative reactions… and lots of colourful figures that, from my perspective, looked rather interesting. Eventually, I discovered what was going on: Lili, Mario and their colleagues were writing a chapter for the Methods in Enzymology series about an enzyme that has accompanied Mario through a significant part of his scientific career. So, I decided to do what any responsible furry padawan would do under such circumstances: interview them.
Oreo: Let’s start with the important question. What is AHAS, and why have you spent so many years talking about it?
Mario: AHAS stands for acetohydroxyacid synthase, also known as acetolactate synthase (ALS). It catalyzes the first common step in the biosynthesis of the branched-chain amino acids (BCAAs) valine, leucine, and isoleucine. One particularly interesting feature is that AHAS is present in plants and microorganisms but absent in animals. This makes it a highly selective target for developing molecules capable of inhibiting plants or microorganisms. In fact, AHAS is the target of five major chemical families of commercial herbicides, comprising more than 50 compounds used for weed control. In recent years, however, we have also learned that some of these inhibitors can act on AHAS enzymes from pathogenic microorganisms, opening interesting possibilities for the development of antimicrobial agents.
Oreo: So… is AHAS important because we can block it?
Lili: Partly, yes. But what makes AHAS fascinating is that it turned out to be much more complex than it initially appeared. It requires three cofactors for proper function: ThDP, FAD, and Mg²⁺. It also undergoes side reactions associated with oxidation and reduction processes. Even oxygen can participate indirectly in events that ultimately affect enzyme activity. This has made understanding how herbicides inhibit AHAS much more interesting than we originally expected.
For a long time, one could imagine a herbicide simply as a molecule that enters, binds to the enzyme, and blocks substrate access. We now know that the story for AHAS is considerably more complex. During catalysis, reactive species can be generated and modifications of the FAD and ThDP cofactors can occur. Inhibition can therefore develop progressively, ultimately leading to enzyme inactivation.
Oreo: I admit that’s considerably more interesting than I expected. So what exactly did you do in this new chapter?
Mario: We tried to bring together, in a practical way, many of the procedures required to work experimentally with plant and fungal AHAS enzymes.
The chapter is entitled “Plant and fungal acetohydroxyacid synthases (AHAS): Methods for heterologous expression, kinetic characterization, and structural analysis” and was written together with the MBAB lab members Liliana Cerda-Mejía, Vivian Gavilanes-Flores, in collaboration with Thierry Lonhienne, and Luke Guddat, at the University of Queensland, who lead AHAS research for more than 20 years.
We begin by explaining AHAS structure, catalysis, and inhibition mechanisms, because it is difficult to apply a protocol correctly without understanding what is actually happening to the enzyme. From there, we move into the laboratory. We describe procedures for the heterologous expression and purification of AHAS, methods for studying its enzyme kinetics, continuous and discontinuous assays, procedures for investigating accumulative inhibition by herbicides, methods for examining ThDP degradation and FAD oxidation, and finally protocols for obtaining crystals suitable for studying the enzyme by X-ray crystallography.
Oreo: In other words, is it a kind of manual for someone who wants to start working with AHAS?
Lili: That’s precisely one of the ideas.
There is an enormous amount of information about AHAS in the scientific literature, but the experimental procedures are scattered across studies published over several decades. In addition, this enzyme has some experimental peculiarities that can produce confusing results if you are not aware of them beforehand. For example, AHAS can exhibit a lag phase before reaching maximum activity. The amount of oxygen available during an experiment can also influence what we observe when studying certain herbicides. Even the method used to measure activity needs to be selected according to the experimental question. For that reason, we didn’t want the chapter to be simply a collection of recipes. We tried to explain why each experiment is performed in a particular way and what is happening at the molecular level while it is being carried out.
Oreo: I’ve seen a lot of three-dimensional structures in your work. What does all of this have to do with crystallography?
Mario: Quite a lot.
Crystallography allowed us to literally see how parts of this system work. AHAS structures have helped us understand where its cofactors are located, how the active site is organized, where herbicides bind, and what changes can occur when an inhibitor interacts with the enzyme.
That’s why the chapter concludes with procedures for crystallization and structural analysis. For us, biochemistry, kinetics, and structural biology are not separate stories. They are different ways of looking at the same phenomenon.
Oreo: I have to ask you a less scientific question. I watched you write this chapter for quite a long time and, strangely enough, you seemed to enjoy it. What did this project mean to you?
Mario: That was probably the part I didn’t expect when I accepted the invitation to contribute a chapter on AHAS to Methods in Enzymology. I was initially very excited about the invitation because Methods in Enzymology is one of the leading book series in enzyme science, with a long history of contributions from prominent researchers in the field. Being invited to contribute to the series therefore had considerable professional significance for me.
In addition, there was a very personal reason for accepting this project. Many of the experiments that now appear as protocols in the chapter are experiments that we designed and carried out during my PhD. Thierry worked alongside me in the laboratory throughout much of that process and guided me through many of those experiments. Luke, of course, as our boss and mastermind, played a fundamental role in that work and in my development as a scientist.
As I wrote the protocols, I began to remember what it was actually like to perform those experiments. Preparing buffers. Expressing the protein. Purifying it. Measuring activity. Watching a curve finally begin to make sense. Preparing crystals. Discussing an unexpected result. Coming back the next day and starting all over again.
For a few weeks, writing stopped feeling like writing. At times, I felt as though I were back in the laboratory working with them. And when we finally finished the manuscript, I felt something I hadn’t expected: I was very happy to have completed it, but at the same time, I was a little sad that the experience of writing it had come to an end. Above all, it reminded me of something that has become even clearer with time: I was very fortunate and grateful to have worked with Luke and Thierry and to have been part of such a wonderful group.
Oreo: So this chapter doesn’t just explain how to study AHAS.
Lili: Scientifically, I hope it will be useful to researchers who want to express, purify, characterize, or structurally investigate AHAS. I also hope it will help those studying new herbicides, herbicide resistance, or the potential of this enzyme as an antimicrobial target.
But personally, it represents something more. Scientific papers usually show only the final result: a structure, a graph, a kinetic constant, or a conclusion. They rarely show all the days in the laboratory that made those results possible. This chapter allowed us to do the opposite. To take years of published results and turn them back into experiments.
Oreo: One last question. Are you finally done with this enzyme?
Mario: I don’t think so. There is still much to be done with AHAS. I believe this enzyme has enormous potential as a target for the development of new compounds with herbicidal or antimicrobial activity. Moreover, the structures of bacterial AHAS enzymes remain comparatively underexplored, leaving an exciting field of research open for further investigation. AHAS-inhibiting herbicides have already shown promising antifungal activity. However, their potential against pathogenic bacteria remains much less explored and deserves further investigation.
Oreo’s final remarks
After this interview, I have reached an important conclusion. Science is usually told through publications, numbers, and discoveries. But behind a purified protein, a crystallographic structure, or an activity curve are countless hours of work and, more importantly, the people who shared those hours. Years after an experiment has ended, opening a file, looking at a curve, or writing a protocol can take you back to the place where it all happened.
Our chapter in Methods in Enzymology is available for free for a limited time (26 Sep 2026). Don’t miss the opportunity to check it out! You can access the in press, corrected version here: https://authors.elsevier.com/a/1nZgdHRzCbeEe
Molecular Biology and Applied Biotechnology Lab 