Toward the Reconstitution of a Two-Enzyme Cascade for Resveratrol Synthesis on Potyvirus Particles

Abstract

The highly ordered protein backbone of virus particles makes them attractive candidates for use as enzyme nano-carriers (ENCs). We have previously developed a non-covalent and versatile approach for adhesion of enzymes to virus particles. This approach makes use of z33, a peptide derived from the B-domain of Staphylococcus aureus protein A, which binds to the Fc domain of many immunoglobulins. We have demonstrated that with specific antibodies addressed against the viral capsid proteins (CPs) an 87% coverage of z33-tagged proteins can be achieved on potyvirus particles. 4-coumarate coenzyme A ligase (4CL2) and stilbene synthase (STS) catalyze consecutive steps in the resveratrol synthetic pathway. In this study, these enzymes were modified to carry an N-terminal z33 peptide and a C-terminal 6xHis tag to obtain z4CL2His and zSTSHis, respectively. A protein chimera, z4CL2::STSHis, with the same modifications was also generated from the genetic fusion of both mono-enzyme encoding genes. All z33 enzymes were biologically active after expression in Escherichia coli as revealed by LC-MS analysis to identify resveratrol and assembled readily into macromolecular complexes with Potato virus A particles and α-PVA CP antibodies. To test simultaneous immobilization-purification, we applied the double antibody sandwich – ELISA protocol to capture active z33-containg mono-enzymes and protein chimera directly from clarified soluble cell lysates onto the virus particle surface. These immobilized enzymes were able to synthesize resveratrol. We present here a bottom up approach to immobilize active enzymes onto virus-based ENCs and discuss the potential to utilize this method in the purification and configuration of nano-devices.

Field-Effect Sensors for Virus Detection: From Ebola to SARS-CoV-2 and Plant Viral Enhancers

“…If employed as immobilization scaffolds for biomolecules, from peptides and antibodies up to enzymes, plant VLPs exhibit special advantages (Sapsford et al, 2006;Werner et al, 2006;Comellas-Aragones et al, 2007;Minten et al, 2011;Aljabali et al, 2012;Pille et al, 2013;Uhde-Holzem et al, 2016;Roeder et al, 2017;Dickmeis et al, 2018;Koch et al, 2018b;Tian et al, 2018;Yuste-Calvo et al, 2019a;Aves et al, 2020;Park et al, 2020). This has laid the foundation for novel plant virus-supported biocatalytic nanomaterials (Carette et al, 2007;Cardinale et al, 2012;Koch et al, 2015;Besong-Ndika et al, 2016;Cuenca et al, 2016;Brasch et al, 2017;Schwarz et al, 2017;Aumiller et al, 2018;Chakraborti et al, 2019), and for biodetection formats that may serve as blueprints for novel SARS-CoV-2 sensor layouts, as outlined in the following.…”

Functional Characterization of Pepper Vein Banding Virus-Encoded Proteins and Their Interactions: Implications in Potyvirus Infection

“…The potyvirus-based nanoparticles have also been used as enzyme nano-carriers (ENCs) where the enzymes of the resveratrol synthetic pathway were immobilized on PVA particles. These immobilized enzymes were thus able to synthesize resveratrol [94]. To explore the possibility of PVBV VLPs as nanocarriers, the modes of internalization of PVBV VLPS in different mammalian cells, their intracellular fate and endocytic uptake pathways were studied [95].…”

Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies

“…Up to now, however, the application of viral scaffolds for the presentation of enzymes on their outer surfaces has rarely been investigated, though with promising prospects in most of the few cases published [ 88 , 132 , 152 – 155 ]. By contrast, enzyme encapsulation inside viral cavities is a matter of much broader research [ 72 , 156 – 157 ], in analogy to synthetic polymersomes that are tested extensively for uses as nanoreactors [ 158 – 161 ].…”

“…By these means, a fully active 4-coumarate:CoA-ligase 2 (4CL2; N-terminally fused to Z33) was installed on a distinct filamentous plant virus, zucchini yellow mosaic virus (ZYMV, genus: Potyvirus ), with almost 90% surface coverage [ 88 ]. This work has recently been extended to a related potyvirus, potato virus A (PVA), which could be fashioned with similar surface density not only with 4CL2, but also with a two-enzyme mix of 4CL2 and stilbene synthase (STS) expressed in E. coli, or with a fusion protein of both partner enzymes [ 155 ]. The blended enzymes were able to collaborate in a cascade reaction, converting p -coumaric acid and further precursors into resveratrol.…”

“…This polyphenolic compound is well-known to wine connoisseurs, since it is found predominantly in red grapes and wine and has been extensively discussed to promote health. Although, to date, the fusion protein has not exhibited detectable activity on PVA, and the resveratrol yield from the 4CL2/STS-PVA assemblies was relatively low, the functionality of the enzyme cascade was clearly demonstrated [ 155 ]. Further optimization and transfer to related enzymes might therefore result in biological production platforms for resveratrol or further “functional food” ingredients, employing potato or other plant-virus derived biotemplates as an immobilization matrix.…”