Less Unfavorable Salt Bridges on the Enzyme Surface Result in More Organic Cosolvent Resistance

Abstract

Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However,n ative enzymes often have lowa ctivity and resistance in OSs and at elevated temperatures.H erein, we report as mart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme,B acillus subtilits Lipase A( BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems.I terative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance towardt hree OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and halflife up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50-100 8 8C. The salt bridge redesign provides protein engineers with ap owerful and likely general approach to design OSsand/or thermal-resistant lipases and other a/b-hydrolases.

Capturing a Crucial ‘Disorder-to-Order Transition’ at the Heart of the Coronavirus Molecular Pathology—Triggered by Highly Persistent, Interchangeable Salt-Bridges

“…One common intuition to jam the high-entropy FLCS Spike disordered loop (SARS-CoV-2) into the Furin bound enzyme-substrate complex would be to stabilize the highly localized positive charge cloud electrostatically. It is rather well known that electrostatic interactions play an important role in the dynamic sustenance and transitions associated with protein disorder [111][112][113][114][115][116]. To that end, the suggested electrostatic stabilization of the FLCS Spike would be greatly benefited by the structural proximity of oppositely charged (i.e., anionic) amino acids (coming from Furin) by triggering the formation of Spike-Furin interfacial salt-bridges.…”

“…Such collective dynamics results in salt-bridges of varying persistence and multiplicity across the trajectory, including persistent, as well as transient, salt-bridges [53,54,113,115]. Transient and/or unfavorable salt-bridges have been revealed to be functionally optimized in proteins [53,116] and are often found on enzyme surfaces [116] as well as on strategic locations spread around extended disordered protein regions [53]. For the latter case, one of the evolved key mechanisms towards achieving this functional optimization is to make their charged side chains often amenable to proximally approached ordered protein interactors.…”

Immobilization of Eversa Lipases on Hydrophobic Supports for Ethanolysis of Sunflower Oil Solvent-Free

“…As already described, the stability of the derivatives in anhydrous oil is generally less. To improve the stability of the enzymes studied here, it would be interesting to use [53,54]. Allied to protein engineering techniques is interesting the application of post-immobilization techniques studied in other works by our research group, which investigated immobilizations for similar enzymatic processes [15, 33-35, 55, 56].…”

Capturing a crucial ‘disorder-to-order transition’ at the heart of the coronavirus molecular pathology – triggered by highly persistent, interchangeable salt-bridges

“…One common intuition to jam the high-entropy FLCS Spike disordered loop (SARS-CoV-2) into the Furin bound enzyme-substrate complex would be to stabilize the highly localized positive charge cloud electrostatically. It is rather well known that electrostatic interactions play an important role in the dynamic sustenance and transitions associated with protein disorder [107][108][109][110][111][112]. To that end, the suggested electrostatic stabilization of the FLCS Spike would greatly be benefited by the structural proximity of oppositely charged (i.e., anionic) amino acids (coming from Furin) by triggering the formation of Spike-Furin interfacial salt-bridges.…”

“…Such collective dynamics results in salt-bridges of varying persistence and multiplicity across the trajectory, including persistent as well as transient salt-bridges [49,50,109,111]. Transient and/or unfavorable salt-bridges have been revealed to be functionally optimized in proteins [49,112] and are often found on enzyme surfaces [112] as well as on strategic locations sparsed around extended disordered protein regions [49]. For the later case, one of the evolved key mechanisms towards achieving this functional optimization is to make their charged side chains often amenable to proximally approached ordered protein interactors.…”