Activation of L-lactate oxidase by the formation of enzyme assemblies through liquid-liquid phase separation

Abstract

The assembly state of enzymes is gaining interest as a mechanism for regulating the function of enzymes in living cells. One of the current topics in enzymology is the relationship between enzyme activity and the assembly state due to liquid-liquid phase separation. In this study, we demonstrated enzyme activation via the formation of enzyme assemblies using L-lactate oxidase (LOX). LOX formed hundreds of nanometer-scale assemblies with poly-L-lysine (PLL). In the presence of ammonium sulfate, the LOX-PLL clusters formed micrometer-scale liquid droplets. The enzyme activities of LOX in clusters and droplets were one order of magnitude higher than those in the dispersed state, owing to a decrease in K_M and an increase in k_cat. Moreover, the clusters exhibited a higher activation effect than the droplets. In addition, the conformation of LOX changed in the clusters, resulting in increased enzyme activation. Understanding enzyme activation and assembly states provides important information regarding enzyme function in living cells, in addition to biotechnology applications.

Figure 1

LOX assembly states in the presence of PLL and (NH₄)₂SO₄. (a) Microscopy images of 0.1 µM LOX, 20 mM Tris–HCl (pH 8), 0 or 0.02 mM PLL (concentrations refer to lysine monomer units), 0 or 10 mM (NH₄)₂SO₄. Scale bar, 10 µm (b) DLS data of sample solutions containing 0.1 µM LOX, 0.02 mM PLL, 20 mM Tris–HCl (pH 8), with 0 or 10 mM (NH₄)₂SO₄. (c) Schematic image of assembly states of LOX with PLL and (NH₄)₂SO₄.

Figure 2

Features of LOX-PLL droplets. (a) Bright-field microscopic images of droplets (left) and fluorescent microscopic images of LOX (middle) and PLL-RBITC (right). The solution contained 5 μM LOX, 1 mM PLL, 6 mM (NH₄)₂SO₄, 20 mM Tris–HCl, and 20 mM MES (pH 8). Scale bar, 20 μm. (b) Localization of LOX and PLL-RBITC in the droplet. Fluorescence intensity along the dashed white lines was quantified from the brightness of each pixel. (c) Bright-field microscopic images of coalescing LOX-PLL droplets. The solution contained 5 μM LOX, 1 mM PLL, 6 mM (NH₄)₂SO_4, 20 mM Tris–HCl, and 20 mM MES (pH 8). Scale bar, 10 μm.

Figure 3

Assembly state and activity of LOX depend on the concentration of ammonium sulfate. (a) Bright-field microscopic images of enzyme assemblies. The solution contained 0.1 μM LOX, 0.02 mM PLL, 0–10 mM (NH₄)₂SO₄, and 20 mM Tris–HCl (pH 8). Scale bar, 20 μm. (b) DLS data of sample solution containing 0.1 µM LOX, 0.02 mM PLL, and 20 mM Tris–HCl (pH 8), with 0–10 mM (NH₄)₂SO₄. (c) Enzymatic activity of LOX in the presence of PLL and ammonium sulfate. Relative enzyme activity was defined as the initial reaction velocity in each condition divided by that in the absence of PLL and (NH₄)₂SO₄.

Figure 4

Enzyme kinetics of LOX in clusters_lox. (a) and droplets (b). The sample solution contained 0.1 µM LOX, 0 or 0.02 mM PLL, 0–8 mM L-lactic acid, 20 mM Tris–HCl (pH 8), and 0 or 10 mM (NH₄)₂SO₄.

Figure 5

Far-UV CD spectra of LOX and PLL in clusters_lox. (a) CD spectra of 1 μM LOX (solid line) and 0.2 mM PLL (broken line). (b) CD spectrum of LOX and PLL mixture (solid line), and CD spectrum calculated from that of LOX and PLL in A (broken line). (c) CD spectra of PDLL, LOX and LOX with PDLL.