We describe the design and evolution of catalytic hydrolase activity on a supramolecular protein scaffold, Zn:RIDC1, which was constructed from cytochrome cb building blocks via a metal-templating strategy. Previously, we reported that Zn:RIDC1 could be tailored with tripodal (His/His/Glu), unsaturated Zn coordination motifs in its interfaces to generate a variant termed Zn:AB3, which in turn displayed catalytic activity for the hydrolysis of activated esters and β-lactam antibiotics. Zn:AB3 was subsequently subjected to directed evolution via an in vivo selection strategy, leading to a variant Zn:AB3 which displayed enzyme-like Michaelis-Menten behavior for ampicillin hydrolysis. A criterion for the evolutionary utility or designability of a new protein structure is its ability to accommodate different active sites. With this in mind, we examined whether Zn:RIDC1 could be tailored with alternative Zn coordination sites that could similarly display evolvable catalytic activities. We report here a detailed structural and functional characterization of new variant Zn:AB5, which houses similar, unsaturated Zn coordination sites to those in Zn:AB3, but in completely different microenvironments. Zn:AB5 displays Michaelis-Menten behavior for ampicillin hydrolysis without any optimization. Yet, the subsequent directed evolution of Zn:AB5 revealed limited catalytic improvement, which we ascribed to the local protein rigidity surrounding the Zn centers and the lack of evolvable loop structures nearby. The relaxation of local rigidity via the elimination of adjacent disulfide linkages led to a considerable structural transformation with a concomitant improvement in β-lactamase activity. Our findings reaffirm previous observations that the delicate balance between protein flexibility and stability is crucial for enzyme design and evolution.

We have extracted and categorized the desirable properties of proteins that are adapted as the scaffolds for artificial metalloenzymes.

Graspetides, also known as omega-ester-containing peptides (OEPs), are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) bearing side-to-side macrolactone or macrolactam linkages.Here we present molecular details of the precursor recognition of the macrocyclase enzyme PsnB in the biosynthesis of plesiocin, a Group 2 graspetide. Biochemical analysis revealed that, in contrast to other RiPPs, the core region of the plesiocin precursor peptide noticeably enhanced the enzyme-precursor interaction via the conserved glutamates. We obtained four crystal structures of symmetric or asymmetric PsnB dimers including those with a bound core peptide and a nucleotide, and suggest that the highly conserved Arg213 at the enzyme active site specifically recognizes a ring-forming acidic residue and escorts it to ATP for phosphorylation. Collectively, this study provides insights into the mechanism underlying substrate recognition in the graspetide biosynthesis, and lays a foundation for engineering new variants..