Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of <i>Ideonella sakaiensis</i> PETase and Variants with Improved Stability and Activity

Joho, Yvonne; Vongsouthi, Vanessa; Spence, Matthew A.; Ton, Jennifer; Gomez, Chloe; Tan, Li Lynn; Kaczmarski, Joe A.; Caputo, Alessandro T.; Royan, Santana; Jackson, Colin J.; Ardèvol, Albert

doi:10.1021/acs.biochem.2c00323

Cited by 38 publications

(40 citation statements)

References 84 publications

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“…Rather, it may simply exhibit some level of promiscuity, which from an evolutionary biochemical perspective refers to the coincidental ability of an enzyme to perform side reactions that are physiologically irrelevant and thus not subject to natural selection (44)(45)(46)(47). Although it can be difficult to establish the biological relevance of a given secondary enzymatic activity, many studies have described the co-option of a preexisting promiscuous or minor function as a means by which a novel physiologically relevant enzymatic activity can evolve (48)(49)(50)(51)(52)(53).…”

Section: Discussionmentioning

confidence: 99%

Evolution and Diversification of Carboxylesterase-like [4+2] Cyclases in Aspidosperma and Iboga Alkaloid Biosynthesis

DeMars,

O’Connor

2023

Preprint

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Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as underground metabolites via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

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Section: Discussionmentioning

confidence: 99%

Evolution and Diversification of Carboxylesterase-like [4+2] Cyclases in Aspidosperma and Iboga Alkaloid Biosynthesis

DeMars,

O’Connor

2023

Preprint

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“…We note that there have been multiple elegant computational studies that have helped reveal the mechanisms of PET-degrading enzymes, − more global conformational changes involved in catalysis, , and contributed to associated engineering effort. − However, corresponding studies of the conformational dynamics of this catalytically important tryptophan remain limited. Therefore, to explore the importance of the S214/I218 substitution for PET hydrolytic activity, doubly substituted variants of the strictly conserved histidine and phenylalanine residues in several PET hydrolases, including the mesophilic Bur PL from a Burkholderiales bacterium (H344/F348), and the thermophilic Tf Cut from Thermobifida fusca (H224/F228) and LCC (H218/F222), were generated based on the S214/I218 residues unique to Is PETase.…”

Section: Introductionmentioning

confidence: 99%

Conformational Selection of a Tryptophan Side Chain Drives the Generalized Increase in Activity of PET Hydrolases through a Ser/Ile Double Mutation

et al. 2023

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Poly(ethylene terephthalate) (PET) is the most common polyester plastic in the packaging industry and a major source of environmental pollution due to its single use. Several enzymes, termed PET hydrolases, have been found to hydrolyze this polymer at different temperatures, with the enzyme from Ideonella sakaiensis (IsPETase) having optimal catalytic activity at 30–35 °C. Crystal structures of IsPETase have revealed that the side chain of a conserved tryptophan residue within an active site loop (W185) shifts between three conformations to enable substrate binding and product release. This is facilitated by two residues unique to IsPETase, S214 and I218. When these residues are inserted into other PET hydrolases in place of the otherwise strictly conserved histidine and phenylalanine residues found at their respective positions, they enhance activity and decrease T opt. Herein, we combine molecular dynamics and well-tempered metadynamics simulations to investigate dynamic changes of the S214/I218 and H214/F218 variants of IsPETase, as well as three other mesophilic and thermophilic PET hydrolases, at their respective temperature and pH optima. Our simulations show that the S214/I218 insertion both increases the flexibility of active site loop regions harboring key catalytic residues and the conserved tryptophan and expands the conformational plasticity of this tryptophan side chain, enabling the conformational transitions that allow for substrate binding and product release in IsPETase. The observed catalytic enhancement caused by this substitution in other PET hydrolases appears to be due to conformational selection, by capturing the conformational ensemble observed in IsPETase.

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“…Recently a number of exciting articles have been published reporting improved catalytic activities of either IS, PET2, or LCC. 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 A variety of strategies targeting sequence‐based knowledge, substrate‐binding or the catalytic mechanism have been used including machine‐learning or directed evolution that resulted in enhanced degradation capabilities thereby emphasizing the high potential of engineered PET‐hydrolases. In fact, Tournier et al already presented a proof of concept for enzymatic PET recycling employing engineered variants of LCC.…”

Section: Introductionmentioning

confidence: 99%

“…Over the last years, protein engineering has been applied to increase activity and thermostability of these enzymes, enabling future recycling applications. Recently a number of exciting articles have been published reporting improved catalytic activities of either IS, PET2, or LCC 12‐19 . A variety of strategies targeting sequence‐based knowledge, substrate‐binding or the catalytic mechanism have been used including machine‐learning or directed evolution that resulted in enhanced degradation capabilities thereby emphasizing the high potential of engineered PET‐hydrolases.…”

Section: Introductionmentioning

confidence: 99%

Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes

et al. 2022

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The handling of plastic waste and the associated ubiquitous occurrence of microplastic poses one of the biggest challenges of our time. Recent investigations of plastic degrading enzymes have opened new prospects for biological microplastic decomposition as well as recycling applications. For polyethylene terephthalate, in particular, several natural and engineered enzymes are known to have such promising properties. From a previous study that identified new PETase candidates by homology search, we chose the candidate PET6 from the globally distributed, halophilic organism Vibrio gazogenes for further investigation. By mapping the occurrence of Vibrios containing PET6 homologs we demonstrated their ubiquitous prevalence in the pangenome of several Vibrio strains. The biochemical characterization of PET6 showed that PET6 has a comparatively lower activity than other enzymes but also revealed a superior turnover at very high salt concentrations. The crystal structure of PET6 provides structural insights into this adaptation to saline environments. By grafting only a few beneficial mutations from other PET degrading enzymes onto PET6, we increased the activity up to three‐fold, demonstrating the evolutionary potential of the enzyme. MD simulations of the variant helped rationalize the mutational effects of those mutants and elucidate the interaction of the enzyme with a PET substrate. With tremendous amounts of plastic waste in the Ocean and the prevalence of Vibrio gazogenes in marine biofilms and estuarine marshes, our findings suggest that Vibrio and the PET6 enzyme are worthy subjects to study the PET degradation in marine environments.

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Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity

Cited by 38 publications

References 84 publications

Evolution and Diversification of Carboxylesterase-like [4+2] Cyclases in Aspidosperma and Iboga Alkaloid Biosynthesis

Evolution and Diversification of Carboxylesterase-like [4+2] Cyclases in Aspidosperma and Iboga Alkaloid Biosynthesis

Conformational Selection of a Tryptophan Side Chain Drives the Generalized Increase in Activity of PET Hydrolases through a Ser/Ile Double Mutation

Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes

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