Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4

Kodai Kurihara, Koji Umezawa, Ann E. Donnelly, Brendan Sperling, Guanyu Liao, Michael H. Hecht, Ryoichi Arai

Research output: Contribution to journalArticlepeer-review

Abstract

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Previously, we reported Syn-F4, the first de novo protein that meets both criteria. Syn-F4 hydrolyzed the siderophore ferric enterobactin, and expression of Syn-F4 allowed an inviable strain of Escherichia coli (Δfes) to grow in iron-limited medium. Here, we describe the crystal structure of Syn-F4. Syn-F4 forms a dimeric 4-helix bundle. Each monomer comprises two long α-helices, and the loops of the Syn-F4 dimer are on the same end of the bundle (syn topology). Interestingly, there is a penetrated hole in the central region of the Syn-F4 structure. Extensive mutagenesis experiments in a previous study showed that five residues (Glu26, His74, Arg77, Lys78, and Arg85) were essential for enzymatic activity in vivo. All these residues are located around the hole in the central region of the Syn-F4 structure, suggesting a putative active site with a catalytic dyad (Glu26–His74). The complete inactivity of purified proteins with mutations at the five residues supports the putative active site and reaction mechanism. Molecular dynamics and docking simulations of the ferric enterobactin siderophore binding to the Syn-F4 structure demonstrate the dynamic property of the putative active site. The structure and active site of Syn-F4 are completely different from native enterobactin esterase enzymes, thereby demonstrating that proteins designed de novo can provide life-sustaining catalytic activities using structures and mechanisms dramatically different from those that arose in nature.

Original languageEnglish (US)
Article numbere2218281120
JournalProceedings of the National Academy of Sciences of the United States of America
Volume120
Issue number38
DOIs
StatePublished - Sep 19 2023

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • binary patterned library
  • de novo enzyme
  • de novo protein
  • dimeric 4-helix bundle
  • ferric enterobactin esterase

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