8ZYG

Crystal structure of a cupin protein (tm1459, I49C-4py/H52A/C106D mutant) in copper (Cu) substituted form

Summary for 8ZYG

• Entry DOI: 10.2210/pdb8zyg/pdb
• Descriptor: Cupin type-2 domain-containing protein, HEXAETHYLENE GLYCOL, COPPER (II) ION, ... (5 entities in total)
• Functional Keywords: artificial metalloenzymes, chemical modification, metal binding protein
• Biological source: Thermotoga maritima MSB8
• Total number of polymer chains: 2
• Total formula weight: 27393.25

Authors

Morita, Y.,Kubo, H.,Matsumoto, R.,Fujieda, N. (deposition date: 2024-06-17, release date: 2024-09-04, Last modification date: 2025-03-19)

Primary citation

Morita, Y.,Kubo, H.,Matsumoto, R.,Fujieda, N.
A thiopyridine-bound mirror-image copper center in an artificial non-heme metalloenzyme.
J.Inorg.Biochem., 260:112694-112694, 2024

PubMed Abstract: Artificial metalloenzymes, in which a metal complex and protein matrix are combined, have been synthesized to catalyze stereoselective reactions using the chiral environment provided by the protein cavity. Artificial metalloenzymes can be engineered by the chemical modification and mutagenesis of the protein matrix. We developed artificial non-heme metalloenzymes using a cupin superfamily protein (TM1459) with a 4-His tetrad-metal-binding motif. The Cu-bound H52A/C106D mutant with 3-His triad showed a S-enantioselective Michael addition of nitromethane to α,β-unsaturated ketone, 2-aza-chalcone 1. In this study, we demonstrated a chemical modification near the copper-binding site of this mutant to reverse its enantioselectivity. For chemical modification, the amino acid on the Si-face of the binding state of 1 to the copper center was replaced with Cys, followed by reaction with 4,4'-dithiopyridine (4-PDS) to form S-(pyridin-4-ylthio)cysteine (Cys-4py). Cu-bound I49C-4py/H52A/C106D showed reversal of the enantioselectivity from S-form to R-form (ee = 71%, (R)). The effect of steric hindrance of the amino acids at position 49 on enantioselectivity was investigated using I49X/H52A/C106D mutants (X = A, C, I, F, and W). Additionally, chemical modification with 2,2'-dithiopyridine (2-PDS) produced I49-2py/H52A/C106D, which showed lower R-enantioselectivity than I49-4py/H52A/C106D. Among the mutants, the 4py-modification on the Si-face was the most effective in reversing the enantioselectivity. By tuning the Re-face side, the H54A mutation introduced into the I49C-4py/H52A/C106D increased the R-enantioselectivity (ee = 88%, (R)). X-ray crystallography revealed a coordinated structure with ligation of thiopyridine in Cu-bound I49C-4py/H52A/H54A/C106D.

PubMed: 39167879
DOI: 10.1016/j.jinorgbio.2024.112694

Experimental method

X-RAY DIFFRACTION (1.08 Å)