Arold, S. | Search bioRxiv

bioRxiv2026

Hierarchically engineered multi-enzyme nanoreactors for in vitro drug biosynthesis and pathway transplantation into cells

Even though most proteins evolved to function within multi-protein systems such as metabolic networks or signaling pathways, most technical protein applications are based on isolated proteins, for example therapeutic antibodies or industrial enzymes. Reliable methods for the stabilization, in vitro operation and intracellular delivery of multi-protein systems could unlock new applications in green biotechnology, diagnostics, and medical therapy. We demonstrate that the entire violacein biosynthesis pathway, consisting of up to six separately purified enzymes, can be infiltrated into a hierarchically etched MIL-101 (eMIL) metal organic framework. eMIL nanoreactors reshaped pathway dynamics and reaction flows, multiplied violacein yield in vitro and enabled pathway reuse, lyophilization and storage. Moreover, eMIL nanoreactors delivered the six-protein system into mammalian cells, where it produced violacein from cell-provided substrates and cofactors. These findings pave the way for the design of "smart" stimuli-responsive multi-enzyme nanoreactors for biotechnological and medical applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/704820v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@2ce26forg.highwire.dtl.DTLVardef@c5fe1eorg.highwire.dtl.DTLVardef@ae8e06org.highwire.dtl.DTLVardef@1ee4f24_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Mg2+ modulates the activity of hyperpolarization-activated calcium currents in plant cells

Hyperpolarization-activated calcium channels (HACCs) are found in the plasma membrane and tonoplast of many plant cell types where they have an important role in Ca2+-dependent signaling. The unusual gating properties of HACCs in plants, i.e., activation by membrane hyperpolarization rather than depolarization, dictates that HACCs are normally open at physiological hyperpolarized resting membrane potentials (the so called pump or P-state), thus, if not regulated, they would be continuously leaking Ca2+ into cells. In guard cells, HACCs are permeable to Ca2+, Ba2+ and Mg2+, activated by H2O2 and the plant hormone abscisic acid (ABA) and their activity is greatly reduced by low amounts of free cytosolic Ca2+ ([Ca2+]Cyt) and hence will close during [Ca2+]Cyt surges. Here we demonstrate that the presence of the commonly used Mg-ATP inside the cell greatly reduces HACC activity especially at voltages [≤] -200 mV and that Mg2+ causes this block. We therefore conclude, firstly, that physiological cytosolic Mg2+ levels affect HACCs gating and that channel opening requires either high negative voltages ([≥] -200 mV) and/or displacement of Mg2+ away from the immediate vicinity of the channel. Secondly, based on structural comparisons with Mg2+-sensitive animal inward-rectifying K+ channel, we propose that the likely candidate HACCS described here are cyclic nucleotide gated channels (CNGCs), many of which also contain a conserved di-acidic Mg2+-binding motif within their pores. This conclusion is consistent with the electrophysiological data. Finally, we propose that Mg2+, much like in animal cells, is an important component in Ca2+ signalling and homeostasis in plants.

plant biology↗