Investigating the effect of fusion partners on the enzymatic activity and thermodynamic stability of poly(ethylene terephthalate) degrading enzymes.

Plastics are a cornerstone of the modern world, yet the durable material properties that we have come to depend upon have made them recalcitrant environmental pollutants. Biological solutions in the form of engineered enzymes offer low energy and sustainable approaches to recycle and upcycle plastic waste, uncoupling their production and end of life from fossil fuels and greenhouse gases. These enzymes however, encounter immense challenges acting on plastics: facing hydrophobic surfaces, molecular crowding, and high levels of substrate heterogeneity. There have been mixed reports about the benefits of fusing partner domains to polyethylene terephthalate (PET) degrading enzymes, with moderate improvements identified under specific conditions, but no clarity into the factors that underlie the mechanisms. Here, we use the SpyCatcher003:SpyTag003 technology, which demonstrates a profound 47 °C shift in Tm upon irreversible complex formation, to investigate the influence of the thermal stability of the fusion partner on a range of PETases selected for their optimal reaction temperatures. We find that the thermal stability of the fusion partner does not have a positive correlation on the activity of the enzymes or their evident kinetic and thermal stabilities. Instead, it appears that the fusion to less stable SpyCatcher003 tends to increase the measured activation energy of unfolding compared to the more stable complex and wildtype enzymes. Despite this, the fusions to SpyCatcher003 do not show significantly better catalytic activity on PET films, with or without SpyTag003, and were found to be sometimes disruptive. The approach we highlight here, in using a fusion partner with controllable melting temperature, allowed us to dissect the impact of the stability of a fusion partner on enzyme properties. Although fusion stability did not appear to be coupled with identifiable trends in enzymatic activities, careful analysis of the unfolding pathways, and solid and solution activities of a wider range of enzymes may yield a more detailed understanding.

Concentration-Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering.

Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.

Electrochemical and structural coupling of the naphthoquinone amino acid.

As a prelude to engineering artificial energy conversion proteins emulating biology, we examine the inclusion of a synthetic naphthoquinone amino acid in a characterized host-guest protein and determine the effects of its quinone and hydroquinone forms on the helix-coil distribution.

Reversible proton coupled electron transfer in a peptide-incorporated naphthoquinone amino acid.

The synthesis of a naphthoquinone amino acid and its electrochemical characterization in a peptide is presented.

Synthetic hydrogenases: incorporation of an iron carbonyl thiolate into a designed peptide.

[FeFe] hydrogenases catalyze reversible hydrogen oxidation at an unusual organometallic active site. Neither enzymatic studies nor synthesis of small molecule models has managed to elucidate the mechanisms of these enzymes. In this paper, we demonstrate the incorporation of an iron carbonyl thiolate mimic of the hydrogenase active site into a de novo artificial peptide, creating the first peptide-based model system for hydrogenases.

Simultaneous and stereoselective formation of planar and axial chiralities in enantiopure sulfinyl iron diene complexes.

[reaction: see text] Enantiopure (1Z,3E)-1-sulfinyl dienes bearing an o-dithianylphenyl group can be prepared and complexed with (bda)Fe(CO)(3) to afford the corresponding sulfinyl diene iron(0) tricarbonyl complexes. This diastereoselective complexation introduces planar and axial chirality simultaneously, with a high degree of facial selectivity as well as atropselectivity. Dynamic kinetic resolution is likely to be the origin of the atropselectivity.