Catch-and-Release: The Assembly, Immobilisation and Recycling of Redox-reversible Artificial Metalloenzymes

TY - JOUR

T1 - Catch-and-Release

T2 - The Assembly, Immobilisation and Recycling of Redox-reversible Artificial Metalloenzymes

AU - Miller, Alex H

AU - Blagova, Elena V

AU - Large, Benjamin

AU - Booth, Rosalind L

AU - Wilson, Keith S

AU - Duhme-Klair, Anne-K

N1 - © 2024 The Authors. Published by American Chemical Society.

PY - 2024/3/1

Y1 - 2024/3/1

N2 - Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilisation of these bio-hybrid catalysts on support materials to enhance stability and enable their retention, recovery and reuse. Here we describe the immobilisation of polyhistidine-tagged ArMs that allow the redox-controlled replacement of catalytic cofactors that have lost activity, for example due to poisoning or decomposition, on immobilised metal affinity chromatography (IMAC) resins. By using periplasmic siderophore-binding protein scaffolds that originate from thermophilic bacteria (GstCeuE and PthCeuE) in combination with a siderophore-linked imine reduction catalyst, reaction rates were achieved that are about 3.5 times faster than those previously obtained with CjCeuE, the analogous protein of Campylobacter jejuni. Upon immobilisation, the GstCeuE-derived ArM showed a decrease in turnover frequency in the reduction of dehydrosalsolidine by 3.4-fold, whilst retaining enantioselectivity (36%) and showing improved stability that allowed repeat recovery and recycling cycles. Catalytic activity was preserved over the initial four cycles. In subsequent cycles a gradual reduction of activity was evident. Once the initial activity decreased to around 40% of the initial activity (23rd recycling cycle), the redox-triggered artificial cofactor release permitted the subsequent recharging of the immobilised protein scaffold with fresh, active cofactor, thereby restoring the initial catalytic activity of the immobilised ArM and allowing its reuse for several more cycles. Furthermore, the ArM could be assembled directly from protein present in crude cell extracts, avoiding time-consuming and costly protein purification steps. Overall, this study demonstrates that the immobilisation of redox-reversible artificial metalloenzymes facilitates their ‘catch-and-release’ assembly and disassembly and the recycling of their components, improving their potential commercial viability and environmental footprint.

AB - Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilisation of these bio-hybrid catalysts on support materials to enhance stability and enable their retention, recovery and reuse. Here we describe the immobilisation of polyhistidine-tagged ArMs that allow the redox-controlled replacement of catalytic cofactors that have lost activity, for example due to poisoning or decomposition, on immobilised metal affinity chromatography (IMAC) resins. By using periplasmic siderophore-binding protein scaffolds that originate from thermophilic bacteria (GstCeuE and PthCeuE) in combination with a siderophore-linked imine reduction catalyst, reaction rates were achieved that are about 3.5 times faster than those previously obtained with CjCeuE, the analogous protein of Campylobacter jejuni. Upon immobilisation, the GstCeuE-derived ArM showed a decrease in turnover frequency in the reduction of dehydrosalsolidine by 3.4-fold, whilst retaining enantioselectivity (36%) and showing improved stability that allowed repeat recovery and recycling cycles. Catalytic activity was preserved over the initial four cycles. In subsequent cycles a gradual reduction of activity was evident. Once the initial activity decreased to around 40% of the initial activity (23rd recycling cycle), the redox-triggered artificial cofactor release permitted the subsequent recharging of the immobilised protein scaffold with fresh, active cofactor, thereby restoring the initial catalytic activity of the immobilised ArM and allowing its reuse for several more cycles. Furthermore, the ArM could be assembled directly from protein present in crude cell extracts, avoiding time-consuming and costly protein purification steps. Overall, this study demonstrates that the immobilisation of redox-reversible artificial metalloenzymes facilitates their ‘catch-and-release’ assembly and disassembly and the recycling of their components, improving their potential commercial viability and environmental footprint.

U2 - 10.1021/acscatal.3c05294

DO - 10.1021/acscatal.3c05294

M3 - Article

C2 - 38449525

SN - 2155-5435

VL - 14

SP - 3218

EP - 3227

JO - ACS Catalysis

JF - ACS Catalysis

IS - 5

ER -