Engineering substrate specificity into a promiscuous ancestral diterpene synthase

Terpene synthases are a class of enzymes that catalyse the cyclisation of linear unsaturated hydrocarbons into a plethora of cyclic structures, of which many are industrially relevant as fragrances, flavours or due to their medical properties. Despite the structural and functional diversity of their products, members of this enzyme family show a high degree of structural and functional similarity, which is why the rules defining product and substrate specificity are not fully understood. Recently, we have used ancestral sequence reconstruction to design a hypothetical molecular ancestor of spiroviolene synthase - a diterpene synthase from Streptomyces violens that converts the linear C-20 precursor geranylgeranyl pyrophosphate (GGPP) to spiroviolene1. This ancestral enzyme shares 77 % sequence identity with the modern wild-type enzyme from S. violens. Compared to the modern enzyme, the ancestral enzyme showed increased thermostability and an additional new reactivity with the shorter C-15 substrate farnesyl pyrophosphate (FPP) that the modern enzyme did not display. To the best of our knowledge, this is the first time ancestral sequence reconstruction was used on a diterpene cyclase. Based on the ancestral enzyme, a library of enzyme-variants was designed with the aim to influence the substrate specificity of the promiscuous ancestral enzyme. We identified several variants that showed substantial preference for the native substrate GGPP (modern enzyme-like), which demonstrates that the subtle GGPP-preference of the ancestral enzyme could be “evolved” to the GGPP-specific modern enzyme phenotype. Most interestingly, we were also able to identify a few variants that showed reversed substrate preference for FPP over GGPP and thus demonstrated “evolvability” of the ancestor towards the unpreferred shorter substrate. Taken together these findings suggest that the hypothesis of promiscuous evolvable ancestral enzymes might be appropriate for this member of the diterpene synthase family. Moreover, it opens up the exciting prospect of using ancestral sequence reconstruction as a tool to engineer enzyme specificity – either by introducing new desired functionalities next to an already existing one or by reprogramming existing promiscuity towards a desired substrate specificity. Please click Additional Files below to see the full abstract

Rosetta FunFolDes - A general framework for the computational design of functional proteins

The robust computational design of functional proteins has the potential to deeply impact translational research and broaden our understanding of the determinants of protein function and stability. The low success rates of computational design protocols and the extensive in vitro optimization often required, highlight the challenge of designing proteins that perform essential biochemical functions, such as binding or catalysis. One of the most simplistic approaches for the design of function is to adopt functional motifs in naturally occurring proteins and transplant them to computationally designed proteins. The structural complexity of the functional motif largely determines how readily one can find host protein structures that are "designable", meaning that are likely to present the functional motif in the desired conformation. One promising route to enhance the "designability" of protein structures is to allow backbone flexibility. Here, we present a computational approach that couples conformational folding with sequence design to embed functional motifs into heterologous proteins-Rosetta Functional Folding and Design (FunFolDes). We performed extensive computational benchmarks, where we observed that the enforcement of functional requirements resulted in designs distant from the global energetic minimum of the protein. An observation consistent with several experimental studies that have revealed function-stability tradeoffs. To test the design capabilities of FunFolDes we transplanted two viral epitopes into distant structural templates including one de novo "functionless" fold, which represent two typical challenges where the designability problem arises. The designed proteins were experimentally characterized showing high binding affinities to monoclonal antibodies, making them valuable candidates for vaccine design endeavors. Overall, we present an accessible strategy to repurpose old protein folds for new functions. This may lead to important improvements on the computational design of proteins, with structurally complex functional sites, that can perform elaborate biochemical functions related to binding and catalysis

Sequence- and structure guided engineering of proteins and enzymes for biotechnology and health applications

Proteins are highly diverse and sophisticated biomolecules that represent a cornerstone of biological structure and function and have been exploited in man-made applications for thousands of years. Those proteins that facilitate chemical reactions at physiologically relevant time-scales are referred to as enzymes. Understanding the connections between proteins’ functions and their structures, mechanisms and evolution allows to engineer them towards desired properties for various applications. The aim of the work presented in this thesis is to assess different protein engineering approaches and workflows in the context of health and biotechnology applications. Four proteins were studied and/or engineered towards different outcomes using either sequence‑based information, structural information or a combination thereof. In paper I a sequence-based approach was applied to optimise vaccine candidates for severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). Specifically, ancestral sequence reconstruction was used to generate highly stable and soluble antigens that could be produced in high quantities in a low-throughput and structure‑independent manner. These ancestral antigens interacted with antibodies from recovered patients and served as scaffolds to host a domain of the extant antigen to further enhance antibody engagement. Paper II and III applied enzyme engineering to terpene cyclases in a health and biocatalysis context, respectively. In paper II a structure-based approach was used to understand the fundamental principles underlying the catalytic mechanism of an enzyme in human steroid metabolism. Specifically, solvent access tunnels were identified and modified to probe the role of activation entropy in human oxidosqualene cyclase, which drastically modified the temperature dependence of catalysis. This finding may also have implications for engineering related plant enzymes for production of industrially relevant compounds in heterologous hosts. In paper III sequence- and structure based approaches were used together to engineer substrate specificity in a promiscuous bacterial terpene cyclase. Specifically, the structure of a stable reconstructed ancestor of spiroviolene synthase was determined in order to understand the molecular basis of substrate promiscuity and engineer highly selective variants that retained thermostability. The presented workflow is relevant for engineering these enzymes as biocatalysts for production of terpene-based high value compounds. In paper IV the metabolite regulation of a flux-controlling enzyme in the Calvin cycle was studied to eventually engineer it for enhanced growth of autotrophic production hosts. Specifically, interactions between a bifunctional cyanobacterial fructose‑1,6-bisphosphatase and a panel of metabolites were identified using a proteomics approach and verified by in vitro experiments. A synergistic regulation involving the enzyme’s redox state and glyceraldehyde 3‑phosphate was discovered, which has implications for integrated metabolic and enzyme engineering approaches involving this biocatalyst. In summary, the results presented herein highlight the utility of integrating several different engineering approaches for proteins used in health and biotechnology applications. Proteiner är mycket diversa och sofistikerade biomolekyler som representerar en hörnsten för biologisk struktur och funktion och har tagits till vara i tillämpade produkter sen flera tusen år tillbaka. De proteiner som underlättar att kemiska reaktioner händer under en fysiologiskt relevant tidsram kallas för enzymer. En förståelse av sammanhangen mellan proteiners funktion och deras strukturer, mekanismer och evolution möjliggör att utveckla åtråvärda egenskaper hos de olika tillämpningarna.   Målet med det presenterade arbetet i denna avhandling är att granska olika inriktningar och arbetsflöden för att utveckla proteiner med tillämpningar i områdena hälsa och bioteknik.  Fyra proteiner studerades och/eller utvecklades mot olika rön med hjälp av sekvensbaserad information, strukturbaserad information eller en kombination av dessa. I Artikel I tillämpades en sekvensbaserad inriktning för att optimera en vaccinkandidat mot severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, svår akut respiratorisk sjukdom coronavirus 2 på svenska). Konkret, så användes rekonstruktion av förfädersproteiner för att generera mycket stabila och lösliga antigener som kunde produceras i stora mängder. Metoden var inte beroende av att testa många proteiner eller strukturell information. Dessa förfädersantigener interagerade med antikroppar från tillfrisknade patienter och kunde användas som strukturell bas för att hysa en domän som tillhör ett nuvarande antigen med syftet att ytterligare förstärka antikroppsinteraktionerna. I Artikel II och III användes enzymteknik för att utveckla terpencyklaser med tillämpningar inom områdena hälsa respektive biokatalys. I Artikel II tillämpades en strukturbaserad inriktning för att förstå de fundamentala principerna som ligger till grund för en enzymmekanism inom den mänskliga steroid metabolismen. Konkret, så identifierades och modifierades accesstunnlar för vatten med syftet att studera aktiveringsentropins roll för humant oxidosqualencyklas, vilket ledde till en drastisk förändring i katalysens temperaturberoende. Denna insikt kan komma at ha betydelse för utvecklingen av relaterade växtenzymer med syfte att producera industriellt värdefulla kemiska föreninger i cellfabriker. I Artikel III användes sekvensbaserade och strukturbaserade metoder tillsammans för att utveckla substratspecificitet i ett bakteriellt terpencyklas som katalyserar flera reaktioner. Konkret, så löstes strukturen av ett stabilt, rekonstruerat förfädersenzym till spiroviolensyntas för att förstå den molekylära grunden till att enzymet kan katalysera flera reaktioner och för att utveckla mycket selektiva varianter med bibehållen termisk stabilitet. Det presenterade arbetsflödet är relevant för att utveckla dessa enzymer till industriella biokatalysatorer för att producera terpenbaserade kemiska högvärdesföreningar. I Artikel IV studerades hur ett enzym som kontrollerar flödet genom Calvincykeln regleras av metaboliter för att som slutmål utveckla Calvincykeln mot ökad produktion i autotrofiska produktionsvärdar. Konkret, så identifierades interaktionerna mellan ett bifunktionellt fruktos‑1,6-bisfosfatas från cyanobakterier och en utvald grupp metaboliter med hjälp av en proteomikmetod och verifierades sedan med hjälp av in vitro experiment. En synergistisk reglering upptäcktes som involverar enzymets redoxtillstånd och metaboliten glyceraldehyd 3-fosfat och som har konsekvenser för hur detta enzym behöver modifieras för att kunna appliceras inom metabolismteknik. Sammanfattningsvis visar resultaten i denna avhandling nyttan av att integrera flera olika ingenjörsmässiga strategier för att skräddarsy proteiner med tilllämpningar i hälsa och bioteknik. QC 2023-02-27</p

Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase.

Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme's specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology

Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation

Abstract Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering for biotechnology. Here we apply limited proteolysis-small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions shows that some metabolites interact in a species-specific manner. We estimate that approximately 35% of interacting metabolites affect enzyme activity in vitro, and the effect is often minor. Using LiP-SMap data as a guide, we find that the Calvin cycle intermediate glyceraldehyde-3-phosphate enhances activity of fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) from Synechocystis sp. PCC 6803 and Cupriavidus necator in reducing conditions, suggesting a convergent feed-forward activation of the cycle. In oxidizing conditions, glyceraldehyde-3-phosphate inhibits Synechocystis F/SBPase by promoting enzyme aggregation. In contrast, the glycolytic intermediate glucose-6-phosphate activates F/SBPase from Cupriavidus necator but not F/SBPase from Synechocystis. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated

Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation

Metabolite-level regulation of enzyme activity is important for coping with environmental shifts. Recently developed proteomics methodologies allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feedback and feedforward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify metabolite-protein interactions in four Calvin-cycle harboring bacteria, including two cyanobacteria and two chemolithoautotrophs. We identified widespread protein interactions with the metabolites GAP, ATP, and AcCoA in all strains. Some species-specific interactions were also observed, such as sugar phosphates in Cupravidus necator and glyoxylate in Synechocystis sp. PCC 6803. We screened some metabolites with LiP interactions for their effects on kinetics of the enzymes F/SBPase and transketolase, two enzymatic steps of the Calvin cycle. For both Synechocystis and Cupriavidus F/SBPase, GAP showed an activating effect that may be part of feed-forward regulation in the Calvin cycle. While we verified multiple enzyme inhibitors on transketolase, the effect on kinetics was often small. Incorporation of F/SBPase and transketolase regulations into a kinetic metabolic model of Synechocystis central metabolism resulted in a general decreased stability of the network, and altered flux control coefficients of transketolase as well as other reactions. The LiP-SMap methodology is promising for uncovering new modes of metabolic regulation, but will benefit from improved peptide quantification and higher peptide coverage of enzymes, as known interactions are often not detected for low-coverage proteins. . Furthermore, not all LiP interactions appear to be relevant for catalysis, as 4/8 (transketolase) and 5/6 (F/SBPase) of the tested LiP effectors had an effect in in vitroassays.QC 20211117</p

Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation

Metabolite-level regulation of enzyme activity is important for coping with environmental shifts. Recently developed proteomics methodologies allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feedback and feedforward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify metabolite-protein interactions in four Calvin-cycle harboring bacteria, including two cyanobacteria and two chemolithoautotrophs. We identified widespread protein interactions with the metabolites GAP, ATP, and AcCoA in all strains. Some species-specific interactions were also observed, such as sugar phosphates in Cupravidus necator and glyoxylate in Synechocystis sp. PCC 6803. We screened some metabolites with LiP interactions for their effects on kinetics of the enzymes F/SBPase and transketolase, two enzymatic steps of the Calvin cycle. For both Synechocystis and Cupriavidus F/SBPase, GAP showed an activating effect that may be part of feed-forward regulation in the Calvin cycle. While we verified multiple enzyme inhibitors on transketolase, the effect on kinetics was often small. Incorporation of F/SBPase and transketolase regulations into a kinetic metabolic model of Synechocystis central metabolism resulted in a general decreased stability of the network, and altered flux control coefficients of transketolase as well as other reactions. The LiP-SMap methodology is promising for uncovering new modes of metabolic regulation, but will benefit from improved peptide quantification and higher peptide coverage of enzymes, as known interactions are often not detected for low-coverage proteins. . Furthermore, not all LiP interactions appear to be relevant for catalysis, as 4/8 (transketolase) and 5/6 (F/SBPase) of the tested LiP effectors had an effect in in vitroassays.QC 20211117</p

Conformational Selection in Biocatalytic Plastic Degradation by PETase

Due to the steric effects imposed by bulky polymers, the formation of catalytically competent enzyme and substrate conformations is critical in the biodegradation of plastics. In poly(ethylene terephthalate) (PET), the backbone adopts different conformations, gauche and trans, coexisting to different extents in amorphous and crystalline regions. However, which conformation is susceptible to biodegradation and the extent of enzyme and substrate conformational changes required for expedient catalysis remain poorly understood. To overcome this obstacle, we utilized molecular dynamics simulations, docking, and enzyme engineering in concert with high-resolution microscopy imaging and solid-state nuclear magnetic resonance (NMR) to demonstrate the importance of conformational selection in biocatalytic plastic hydrolysis. Our results demonstrate how single-amino acid substitutions in Ideonella sakaiensis PETase can alter its conformational landscape, significantly affecting the relative abundance of productive ground-state structures ready to bind discrete substrate conformers. We experimentally show how an enzyme binds to plastic and provide a model for key residues involved in the recognition of gauche and trans conformations supported by in silico simulations. We demonstrate how enzyme engineering can be used to create a trans-selective variant, resulting in higher activity when combined with an all-trans PET-derived oligomeric substrate, stemming from both increased accessibility and conformational preference. Our work cements the importance of matching enzyme and substrate conformations in plastic hydrolysis, and we show that also the noncanonical trans conformation in PET is conducive for degradation. Understanding the contribution of enzyme and substrate conformations to biocatalytic plastic degradation could facilitate the generation of designer enzymes with increased performance