Biomolecular Condensates Regulate Enzymatic Activity under a Crowded Milieu: Synchronization of Liquid–Liquid Phase Separation and Enzymatic Transformation

Abstract

Cellular crowding plays a key role in regulating the enzymatic reactivity in physiological conditions, which is challenging to realize in the dilute phase. Enzymes drive a wide range of complex metabolic reactions with high efficiency and selectivity under extremely heterogeneous and crowded cellular environments. However, the molecular interpretation behind the enhanced enzymatic reactivity under a crowded milieu is poorly understood. Herein, using the horseradish peroxidase (HRP) and glucose oxidase (GOx) cascade pair, we demonstrate for the first time that macromolecular crowding induces liquid–liquid phase separation (LLPS) via the formation of liquid-like condensates/droplets and thereby increases the intrinsic catalytic efficiencies of HRP and GOx. Both these enzymes undergo crowding induced homotypic LLPS via enthalpically driven multivalent electrostatic as well as hydrophobic interactions. Using a set of kinetic and microscopic experiments, we show that precise synchronization of spontaneous LLPS and enzymatic transformations is key to realize the enhanced enzymatic activity under the crowded environments. Our findings reveal an unprecedented enhancement (91- to 205-fold) in the catalytic efficiency (k cat/K m) of HRP at pH 4.0 within the droplet phase relative to that in the bulk aqueous phase in the presence of different crowders. In addition, we have shown that other enzymes also undergo spontaneous LLPS under macromolecular crowding, signifying the generality of this phenomenon under the crowded environments. More importantly, coalescence driven highly regulated GOx/HRP cascade reactions within the fused droplets have been demonstrated with enhanced activity and specificity under the crowded environments. The present discovery highlights the active role of membraneless condensates in regulating the enzymatic efficacy for complex metabolic reactions under the crowded cellular environments and may find significant importance in the field of biocatalysis.

Macromolecular Crowding Promotes Re-entrant Liquid–Liquid Phase Separation of Human Serum Transferrin and Prevents Surface-Induced Fibrillation

“…17,18,44−46 Chaotropic salt sodium thiocyanate (NaSCN) and aliphatic alcohol 1,6-hexanediol are known to disrupt the hydrophobic protein−protein interactions. 18,44,46 We visualized the feasibility of LLPS of 1 μM Tf in the presence of 10% PEG using CLSM by varying the concentrations of NaSCN from 0.1 to 2.0 M (Figure 3a). Droplet formation is feasible up to 1.0 M of NaSCN; however, complete inhibition of LLPS of Tf has been observed at and beyond 2 M of NaSCN.…”

“…More importantly, our group has recently demonstrated macromolecular crowding-induced biomolecular condensate formation of two structurally robust functional enzymes, namely, horseradish peroxidase (HRP) and glucose oxidase (GOx), which remarkably enhances the enzymatic activity and selectivity at physiological conditions. 46 These intriguing findings of homotypic LLPS of functional proteins open up a new avenue for the exploration of the role of these membraneless condensates to control the activity of various functional proteins. Apart from their role in the aggregation pathway, various synthetic and biomolecular condensates are also known as efficient scaffolds for the stabilization and protection of sequestered biomolecules against temperature, pH, protease digestion, and aging.…”

“…We envisaged that the presence of a distinct LCD might facilitate Tf to undergo phase separation under optimized physiological conditions. However, it should be noted that the presence of IDRs and/or LCDs is not a mandatory prerequisite for biomolecules to undergo LLPS. − To better understand the underlying factors that govern the LLPS and functional aspects of membraneless condensates of biologically active proteins, we have undertaken the present study. In particular, we want to know whether Tf undergoes LLPS and subsequent aggregation via liquid-like condensate formation within the cellular lifetime of Tf under physiological conditions.…”

Biomolecular Liquid–Liquid Phase Separation for Biotechnology

“…Recent studies suggest that some enzymes show different activity inside droplets. For example, Saini et al recently discovered that macromolecular crowding induces LLPS, which leads to an increase in the intrinsic catalytic efficiencies of horseradish peroxidase (HRP) and glucose oxidase (GOx) [ 110 ]. Transcription factors (TFs) and RNAs also induce the formation of transcriptional condensates via LLPS, which contain clusters of multiple enhancers (super-enhancers) [ 111 ].…”

“…To form transcriptional condensates, TFs bind to various cis-regulatory DNA elements (e.g., promoters and enhancers) and stimulate the transcription of active genes in proximity, facilitating the precise control of gene expression [ 113 ]. Other examples of enzymes and transcription factors which undergo LLPS are listed in Table 4 [ 110 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 ].…”