Evolution of a highly active and enantiospecific metalloenzyme from short peptides

Abstract

Primordial sequence signatures in modern proteins imply ancestral origins tracing back to simple peptides. Although short peptides seldom adopt unique folds, metal ions might have templated their assembly into higher-order structures in early evolution and imparted useful chemical reactivity. Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (kcat/KM~ 106M−1s−1). The simultaneous optimization of structure and function in a naïve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.

Primordial emergence of a nucleic acid binding protein via phase separation and statistical ornithine to arginine conversion

“…We thus lack knowledge as to what meaningful functions simple precursor polypeptides that lack a defined 3D structure could fulfill. Polypeptides, and even short peptides, with various biochemical functions have been described 12,15,18 , but the functions described so far have limited evolutionary relevance. Secondly, given a simple polypeptide with a rudimentary yet evolutionary-relevant function, how can this polypeptide gradually evolve to give a contemporary protein with a proficient and specific function?…”

“…Insights as to how a folded, biochemically-active protein can emerge from a simpler polypeptide are rare [16][17][18] , certainly when it comes to the natural protein world. Firstly, biochemical functions such as binding of small ligands demand a globular protein with a preorganized active site.…”

Katalyse und Elektronentransfer in helikalen De‐novo‐Gerüststrukturen

“…MID1‐Zn zeigte gegenüber unserem TRI‐Gerüst eine verbesserte pNPA‐Hydrolyseaktivität mit k cat / K M von 660 m −1 s −1 bei pH 9, was auf die einfachere Zugänglichkeit des aktiven Zentrums im Homodimer zurückgeführt wurde. Vor kurzem beschrieb diese Gruppe die künstliche Evolution des MID1‐Katalysators, wodurch dessen Aktivität in der Hydrolyse eines racemischen fluorogenen Esters um das 70000‐Fache verbessert wurde . Die Gruppe um Tezcan berichtete vom Design einer Metallo‐β‐lactamase bestehend aus einem Tetramer von modifiziertem Cytochrom cb5662, Zn 8 :AB3 4 .…”

A computational method for design of connected catalytic networks in proteins

“…[1][2][3][4][5] Several approaches have been developed to generate new enzyme active sites by searching for placements of catalytically competent side-chain constellations in selected protein scaffolds or curated subsets of the Protein Data Bank containing up to several thousand protein structures. 12 While directed evolution has succeeded in maturing computational designs to have activities comparable to native enzymes, [13][14][15][16][17][18][19] the activities of the original computational designs have generally been quite low. RosettaMatch is then used to search for geometrically compatible placements of these ideal active sites in protein scaffolds.…”

“…RosettaMatch is then used to search for geometrically compatible placements of these ideal active sites in protein scaffolds. 12 While directed evolution has succeeded in maturing computational designs to have activities comparable to native enzymes, [13][14][15][16][17][18][19] the activities of the original computational designs have generally been quite low. Achieving high catalytic activity directly from computation is an outstanding current challenge.…”