8H7E

Crystal structure of a de novo enzyme, ferric enterobactin esterase Syn-F4 (K4T) at 2.0 angstrom resolution

Summary for 8H7E

• Entry DOI: 10.2210/pdb8h7e/pdb
• Related: 8H7C 8H7D
• Descriptor: De novo ferric enterobactin esterase Syn-F4, ACETATE ION (3 entities in total)
• Functional Keywords: binary patterned library, de novo enzyme, dimeric 4-helix bundle, ferric enterobactin esterase, de novo protein
• Biological source: synthetic construct
• Total number of polymer chains: 2
• Total formula weight: 25021.17

Authors

Kurihara, K.,Umezawa, K.,Donnelly, A.E.,Hecht, M.H.,Arai, R. (deposition date: 2022-10-19, release date: 2023-09-20)

Primary citation

Kurihara, K.,Umezawa, K.,Donnelly, A.E.,Sperling, B.,Liao, G.,Hecht, M.H.,Arai, R.
Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4.
Proc.Natl.Acad.Sci.USA, 120:e2218281120-e2218281120, 2023

PubMed 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 (Δ) 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 ( 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.

PubMed: 37695900
DOI: 10.1073/pnas.2218281120

Experimental method

X-RAY DIFFRACTION (2 Å)