Engineering a Covalent Linkage into a Dimeric De Novo Enzyme Reveals a Novel Life-Sustaining Mechanism

Abstract

Designing novel proteins that share no homology with natural sequences, but which nonetheless provide life sustaining functions, is an important goal for synthetic biology. Towards this goal, we previously reported Syn-F4, the first de novo enzyme capable of catalyzing a life-sustaining reaction, both in vitro and in vivo. Syn-F4 catalyzes hydrolysis of the siderophore, ferric enterobactin, thereby releasing iron and enabling growth in iron-limited media of an otherwise inviable {Delta}fes strain of Escherichia coli. Although Syn-F4 provides a direct enzymatic replacement of the natural ferric enterobactin esterase encoded by Fes, it has a dramatically different structure and enzymatic mechanism than the natural Fes enzyme. The novel Syn-F4 enzyme forms a 4-helix bundle, comprising a homodimer of two -helical hairpins. Here we describe the engineering of a covalent peptide linkage into the homodimer to generate a single chain 4-helix bundle. As expected, the resulting linked protein (Syn-F4-Link) also rescued {Delta}fes cells in iron-limited media. Moreover, X-ray crystallography revealed a 3D structure similar to the parental homodimer. Surprisingly, however, the linked protein was not enzymatically active. Instead, Syn-F4-Link rescues {Delta}fes cells by upregulating biosynthesis of the enterobactin siderophore thereby enabling assimilation of sufficient iron to sustain cell growth. These findings demonstrate that two very similar de novo proteins can sustain cell growth using dramatically different biological mechanisms.