Inversion of Enzymatic Enantiocatalysis Mediated by Biomolecular Condensates

Area of Science:

• Biochemistry
• Enzyme kinetics
• Supramolecular chemistry

Background:

• Enzyme enantioselectivity is crucial in biological systems.
• Biocondensates, such as protein and DNA-based assemblies, create unique microenvironments.
• Understanding enzyme behavior within these condensed phases is key for synthetic biology.

Purpose of the Study:

• To investigate the enantioselective phosphodiesterase activity of alkaline phosphatase (ALP).
• To compare ALP enantioselectivity in aqueous buffer, protein biocondensates (BSA), and DNA coacervates.
• To elucidate the role of enzyme conformational dynamics in altered enantioselectivity within biocondensates.

Main Methods:

• Utilized R- and S-forms of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNPP) as RNA-model substrates.
• Enzyme assays were performed in aqueous buffer, bovine serum albumin (BSA) condensates, and DNA complex coacervates.
• Measured enantiomeric excess (E) values to quantify enantioselectivity.
• Analyzed enzyme conformational dynamics using biophysical techniques (implied).

Main Results:

• Native ALP exhibits preference for S-HPNPP in aqueous buffer (E ≈ 33%).
• This S-HPNPP preference is maintained in BSA condensates (E range: 10% to 34%).
• A complete reversal of enantioselectivity is observed in DNA coacervates, with a preference for R-HPNPP (E range: -20% to -47%).
• Reversal of enantioselectivity correlates with changes in ALP's conformational dynamics within the DNA coacervate.

Conclusions:

• Enzyme enantioselectivity can be significantly altered by the microenvironment of biocondensates.
• DNA coacervates induce a conformational change in ALP, leading to reversed substrate preference.
• Findings provide critical insights into enzyme promiscuity and chiral selectivity in engineered cellular environments.
• This research has implications for the design and function of synthetic protocells.