The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase

This article has been Reviewed by the following groups

Read the full article See related articles

Listed in

Log in to save this article


A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active Sc LPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the wild-type enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using SAXS, NMR, MD simulations, and functional assays. The resulting data revealed that, in the case of Sc LPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the wild-type and a serine-rich variant, which have the same linker length, demonstrated better performance compared to those with either a shorter or longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, while the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass processing enzymes is more complex than mere structural tethering.

Article activity feed

  1. Thank you for this amazing publication! I loved your paper! It's so cool how deeply you went to understand the function of a linker sequence using a variety of biophysical and computational techniques. Big kudos! I personally find it fascinating how a short amino acid sequence, that's technically not part of the core enzyme, could have such a profound impact on the main function of said protein! Specifically in your case, it seems the linker impacts catalytic activity as well as thermal stability of the enzyme. Also it seems that the linker may be impacting substrate binding. You speculate on this in the discussion but I was wondering if I could probe a little further. First, how much is known about the interaction of this enzyme with a complex substrate such as cellulose? For example, is this LPMO, or this class in general, a processive enzyme? Does the enzyme always remain bound to the substrate throughout catalysis? Or does it detach, and then re-attach? Second, is it known how LPMOs locate the scissile bond within the complex cellulose substrate? For example, do they attach at a random place on the substrate and then begin searching for the scissile bond (search in 2-D space)? Or do they search for the bond by attaching and detaching numerous times across the substrate (search in 3-D space)? Thank you so much for the cool paper! And thank you for your time!