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Enzymes have multiple applications in medicine but during the past decades interest in the application of enzymes as (bio)catalysts to produce a wide range of valuable molecules in various industries has increased. Many chemical compounds (from pharmaceuticals to bulk commodities) can be produced by a series of enzymatically-catalysed chemical steps, and in many cases one of these steps is an oxidation. The use of molecular oxygen as an oxidising agent in biocatalytic processes is a double-edged approach. From one side, the oxygen is supplied to the reactor in the form of air bubbling, which is cheap, highly available and non-toxic. From the other side, bubbling air into the reaction media creates a gas-liquid interface which adsorbs enzymes and compromises their stability. Moreover, the oxygen is quite insoluble in water, which often results in oxygen-limited reactions. These aspects are the main limiting factors for the stability and kinetics of enzymes that perform oxidative biocatalysis and prevent the reaction from happening at a rate that is high/competitive enough for industrial feasibility. Therefore, we need systems to mimic and understand better these factors to try and mitigate their effects upon scale-up. In this review, we present two complementary systems to study these factors: one apparatus that ensures a constant gas-liquid interface and another one that maintains a constant oxygen partial pressure. Both can provide highly valuable information regarding the maximum rate of reaction and about the deactivation profiles of enzymes in the presence of bubbles.

Prolyl oligopeptidases from psychrophilic, mesophilic, and thermophilic organisms found in a range of natural environments are studied using a combination of protein structure prediction, atomistic molecular dynamics, and trajectory analysis to determine how the S9 protease family adapts to extreme thermal conditions. We compare our results with hypotheses from the literature regarding structural adaptations that allow proteins to maintain structure and function at extreme temperatures, and we find that, in the case of prolyl oligopeptidases, only a subset of proposed adaptations are employed for maintaining stability. The catalytic and propeller domains are highly structured, limiting the range of mutations that can be made to enhance hydrophobicity or form disulfide bonds without disrupting the formation of necessary secondary structure. Rather, we observe a pattern in which overall prevalence of bound interactions (salt bridges and hydrogen bonds) is conserved by using increasing numbers of increasingly short-lived interactions as temperature increases. This suggests a role for an entropic rather than energetic strategy for thermal adaptation in this protein family.

Biocatalysis is the use of enzymes to accelerate chemical reactions and has undeniable advantages over conventional catalysis, since it significantly reduces the use of toxic compounds and solvents, generates less hazardous waste, and allows working in less aggressive conditions of temperature and pH, Despite these benefits, enzymes due to their protein nature can be easily denatured and being soluble, they are not recoverable from the reaction medium, therefore, enzymatic immobilization in a specialty of biocatalysis in constant development and growth since it allows a greater stability, reuse and easy recovery of the medium, its use is already widespread and is increasing in industries such as pharmaceuticals, biofuels, the food industry and the production of flavors, fragrances and cosmetics. The different strategies to obtain immobilized derivatives are addressed in this document., La biocatálisis es el uso de enzimas para acelerar las reacciones químicas y posee innegables ventajas sobre la catálisis convencional, ya que disminuye sensiblemente el uso de compuestos tóxicos y disolventes, genera menos residuos peligrosos, y permite trabajar en condiciones menos agresivas de temperatura y pH, a pesar de estas bondades, las enzimas, por su naturaleza proteica, pueden ser fácilmente desnaturalizadas y al ser solubles, no son recuperables del medio de reacción, por ello, la inmovilización enzimática es una especialidad de la biocatálisis en constante desarrollo y crecimiento ya que permite una mayor estabilidad, reuso y fácil recuperación del medio, su aplicación es ya generalizada y va en aumento en industrias tales como la farmacéutica, la de los biocombustibles, la alimentaria y de producción de sabores, fragancias y cosméticos. Las diferentes estrategias para obtener derivados inmovilizados se abordan en el presente documento.

The lipases from Thermomyces lanuginosus (TLL) and Candida antarctica (B) (CALB) were immobilized on octyl-agarose beads at 1 mg/g (a loading under the capacity of the support) and by overloading the support with the enzymes. These biocatalysts were compared in their stabilities in 10 mM of sodium phosphate, HEPES, and Tris-HCl at pH 7. Lowly loaded CALB was more stable than highly loaded CALB preparation, while with TLL this effect was smaller. Phosphate was very negative for the stability of the CALB biocatalyst and moderately negative using TLL at both loadings. The stability of the enzymes in HEPES and Tris-HCl presented a different response as a function of the enzyme loading (e.g., using lowly loaded CALB, the stabilities were similar in both buffers, but it was clearly smaller in HEPES using the highly loaded biocatalysts). Moreover, the specific activity of the immobilized enzymes versus p-nitrophenol butyrate, triacetin and R- or S-methyl mandelate depended on the buffer, enzyme loading, and interaction between them. In some cases, almost twice the expected activity could be obtained using highly loaded octyl-CALB, depending on the buffer. A co-interaction between the effects on enzyme activity and the specificity of support enzyme loading and buffer nature was detected.

In recent decades, there has been a growing emphasis on improving investigation capacities in the field of forensic toxicology. This study looks into the interaction between acetylcholinesterase (AChE) and glyphosate, specifically its potential suppression. The study optimizes parameters such as pH levels, enzyme concentrations, and substrate concentrations using the Ellman enzymatic assay. AChE activity is best maintained at 50 mU with 3 mM DTNB and 5 mM ACth, and storage at 4°C assures stability. The study also exposes glyphosate's inhibitory effect on AChE, demonstrating its potential hazards to human health and the environment. Comparisons with butyrylcholinesterase (BChE) demonstrate each enzyme's distinct stability and selectivity. These findings shed light on the complex interactions between glyphosate and cholinesterase, paving the path for breakthroughs in toxicology and related sciences., Embargo status: Restricted until 01/2027. To request the author grant access, click on the PDF link to the left.

De novo enzyme design has sought to introduce active sites and substrate-binding pockets that are predicted to catalyse a reaction of interest into geometrically compatible native scaffolds1,2, but has been limited by a lack of suitable protein structures and the complexity of native protein sequence-structure relationships. Here we describe a deep-learning-based 'family-wide hallucination' approach that generates large numbers of idealized protein structures containing diverse pocket shapes and designed sequences that encode them. We use these scaffolds to design artificial luciferases that selectively catalyse the oxidative chemiluminescence of the synthetic luciferin substrates diphenylterazine3 and 2-deoxycoelenterazine. The designed active sites position an arginine guanidinium group adjacent to an anion that develops during the reaction in a binding pocket with high shape complementarity. For both luciferin substrates, we obtain designed luciferases with high selectivity; the most active of these is a small (13.9 kDa) and thermostable (with a melting temperature higher than 95 °C) enzyme that has a catalytic efficiency on diphenylterazine (kcat/Km = 106 M-1 s-1) comparable to that of native luciferases, but a much higher substrate specificity. The creation of highly active and specific biocatalysts from scratch with broad applications in biomedicine is a key milestone for computational enzyme design, and our approach should enable generation of a wide range of luciferases and other enzymes.

A key aspect of adaptation to cold environments is the production of cold-active enzymes by psychrophilic organisms. These enzymes not only have high activity at low temperatures, but also exhibit remarkable structural flexibility and thermolability. In this context, the role of metal ions has been little explored, and the few available studies seem to suggest that metal binding counteracts structural flexibility. This article reports an investigation into the role of the binding of manganese ion (Mn2+) in the thermal adaptation of an esterase (M-Est) of the GDSx family, identified in the genome of the Antarctic bacterium Marinomonas sp. ef1. M-Est is specific for esters containing acetate groups and turned out to be a highly thermolabile cold-active enzyme, with a catalysis optimum temperature of 5 °C and a melting temperature of 31.7 °C. A combination of biochemical and computational analyses, including molecular dynamics simulations, revealed that M-Est binds Mn2+ ions via a single binding site located on the surface of the enzyme, close to the active site. Although the interaction between M-Est and Mn2+ induces only local conformational changes involving the active site, quite surprisingly they trigger an improvement in both thermal stability and catalytic efficiency under mild temperature conditions. These results, together with the conservation of the Mn2+ binding site among psychrophilic and psychrotolerant homologues, suggest that Mn2+ binding may be a useful, albeit atypical, strategy to mitigate the detrimental effects of temperature on true cold-active enzymes.

Biocatalytic oxidation is an interesting prospect for the selective synthesis of active pharmaceutical intermediates. Bubbling air or oxygen is considered as an efficient method to increase the gas–liquid interface and thereby enhance oxygen transfer. However, the enzyme is deactivated in this process and needs to be further studied and understood to accelerate the implementation of oxidative biocatalysis in larger production processes. This paper reports data on the stability of NAD(P)H oxidase (NOX) when exposed to different gas–liquid interfaces introduced by N2 (0% oxygen), air (21% oxygen), and O2 (100% oxygen) in a bubble column. A pH increase was observed during gas bubbling, with the highest increase occurring under air bubbling from 6.28 to 7.40 after 60 h at a gas flow rate of 0.15 L min–1. The kinetic stability of NOX was studied under N2, air, and O2 bubbling by measuring the residual activity, the deactivation constants (kd1) were 0.2972, 0.0244, and 0.0346 with the corresponding half-lives of 2.2, 28.6, and 20.2 h, respectively. A decrease in protein concentration of the NOX solution was also observed and was attributed to likely enzyme aggregation at the gas–liquid interface. Most aggregation occurred at the air–water interface and decreased greatly from 100 to 14.16% after 60 h of bubbling air. Furthermore, the effect of the gas–liquid interface and the dissolved gas on the NOX deactivation process was also studied by bubbling N2 and O2 alternately. It was found that the N2–water interface and O2–water interface both had minor effects on the protein concentration decrease compared with the air–water interface, whilst the dissolved N2 in water caused serious deactivation of NOX. This was attributed not only to the NOX unfolding and aggregation at the interface but also to the N2 occupying the oxygen channel of the

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme®435 (L435) (lipase B from Candida antarctica), Lipozyme®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process., Ministerio de Ciencia e Innovación (MICINN), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil, Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Fac. de Ciencias Biológicas, TRUE, pub

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme®435 (L435) (lipase B from Candida antarctica), Lipozyme®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process., Ministerio de Ciencia e Innovación (MICINN), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil, Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Fac. de Ciencias Biológicas, TRUE, pub

Transaminases are pyridoxal 5’-phosphate (PLP)-dependent enzymes that transfer amino-functions. The transaminase from Silicibacter pomeroyi (SpATA) exhibits a broad substrate spectrum. In this work we examined the effect of different conditions (light, buffer and PLP-concentration) on the stability of SpATA, as well as the causes for these effects. The enzyme was stored either in TRIS or CHES with 0–10 mM added PLP at 22 °C. The samples were either kept dark or they were exposed to light. The results show that invariably, all samples kept in darkness exhibited longer half-life times than the ones exposed to light. An increase in the half-life from 8 h to 720 h could be achieved solely by keeping the sample dark. Especially samples in CHES buffer inactivated faster in light the more PLP was present, due to the degradation of PLP. In TRIS however, an imine-bond between TRIS and PLP protects PLP from degradation., QC 20221221, Horizon 2020 MSCA INTERfaces

Enzymes have several excellent catalytic features, and the last few years have seen a revolution in biocatalysis, which has grown from using one enzyme to using multiple enzymes in cascade reactions, where the product of one enzyme reaction is the substrate for the subsequent one. However, enzyme stability remains an issue despite the many benefits of using enzymes in a catalytic system. When enzymes are exposed to harsh process conditions, deactivation occurs, which changes the activity of the enzyme, leading to an increase in reaction time to achieve a given conversion. Immobilization is a well-known strategy to improve many enzyme properties, if the immobilization is properly designed and controlled. Enzyme co-immobilization is a further step in the complexity of preparing a biocatalyst, whereby two or more enzymes are immobilized on the same particle or support. One crucial problem when designing and using co-immobilized enzymes is the possibility of using enzymes with very different stabilities. This paper discusses different scenarios using two co-immobilized enzymes of the same or differing stability. The effect on operational performance is shown via simple simulations using Michaelis–Menten equations to describe kinetics integrated with a deactivation term. Finally, some strategies for overcoming some of these problems are discussed.

The use of highly selective enzymes to catalyze value-added reactions outside the cell is commonly termed biocatalysis. In this brief perspective, some of the future opportunities for the application of biocatalysis are discussed. First, there are opportunities using multi-enzyme cascades where entirely new synthetic routes can be created independent of cellular constraints. Here the target is mostly high-priced products, such as pharmaceuticals. Secondly, there also exist opportunities for biocatalysis in the synthesis of low-priced products where the high productivities achievable make them eminently suited for drop-in solutions. Both options provide a wealth of interesting research and development possibilities, which are also discussed.

Biocatalysis is a growing discipline, allowing selective catalysis under mild conditions. In particular, protein engineering offers the potential to change enzyme properties and also offers the discovery of entirely new reactions. For implementation in industry, reaction performance metrics (including reaction yield, productivity, product concentration, and specific yield) need to meet minimum targets in order to be cost effective. An important consequence of this is that enzymes need to work under very different conditions from those usually found in nature. Industrial conditions can include high concentrations of substrate and product, as well as the presence of gas-liquid and liquid-liquid interfaces. Conventional enzymology has focused on understanding enzymes under a relatively narrow range of conditions, but the need now is for a new enzymology focused on industrially relevant conditions. In order to achieve this, new scale-down experiments that can mimic industrial conditions at a laboratory scale will be required.

This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47◦C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance., BT/Biocatalysis

Single enzyme nanoparticles (SENs), which encapsulate individual enzymes in a thin permeable polymer network offer great control over the chemical and physical environment directly around the enzyme. SENs have exhibited great enzyme stability by restricting enzyme extensive unfolding motion under extreme conditions, like extreme pH and high temperature. However, up to date, the control over the chemistry of the shell is still quite limited. In this thesis, a new SEN formation strategy has been explored. In order to minimize the risk of enzyme deactivation during synthesis of the SENs, the weak electrostatic interaction was utilized to assemble charged polymers around the enzyme. Different lengths of charged polymers were pre-prepared via reversible addition−fragmentation chain-transfer polymerization (RAFT) and then attached to the surface of enzyme via electrostatic interactions. This strategy has been investigated for the different enzyme, including lysozyme, trypsin, protease, horseradish peroxidase, and glucose oxidase. Isothermal titration calorimetry (ITC) and asymmetric flow field-flow fraction (AF4) in combination with multiangle light scattering (MALS) reveal the binding number and strength of polymer chains / enzyme. The strength of binding can be tuned based on the charge density of the bound polymer. In this method, the trithiocarbonate group of a RAFT agent was placed close to the surface of the enzyme and the initiation of a free radical acrylamide / bisacrylamide polymerisation in solution can result in chain extension of the RAFT polymer and direct the formation of the newly formed hydrogel around the outside of the enzyme. AF4-MALS and small-angle X-ray scattering (SAXS) confirm the formation of a thin cross-linked shell around the enzyme. The mild conditions of this method of SEN formation, which avoids any covalent modification of the enzyme, results in no loss in activity on our model enzyme (glucose oxidase), and four-fold increase in thermal st

Successfully interfacing enzymes and biomachinery with polymers affords on-demand modification and/or programmable degradation during the manufacture, utilization and disposal of plastics, but requires controlled biocatalysis in solid matrices with macromolecular substrates1-7. Embedding enzyme microparticles speeds up polyester degradation, but compromises host properties and unintentionally accelerates the formation of microplastics with partial polymer degradation6,8,9. Here we show that by nanoscopically dispersing enzymes with deep active sites, semi-crystalline polyesters can be degraded primarily via chain-end-mediated processive depolymerization with programmable latency and material integrity, akin to polyadenylation-induced messenger RNA decay10. It is also feasible to achieve processivity with enzymes that have surface-exposed active sites by engineering enzyme-protectant-polymer complexes. Poly(caprolactone) and poly(lactic acid) containing less than 2 weight per cent enzymes are depolymerized in days, with up to 98 per cent polymer-to-small-molecule conversion in standard soil composts and household tap water, completely eliminating current needs to separate and landfill their products in compost facilities. Furthermore, oxidases embedded in polyolefins retain their activities. However, hydrocarbon polymers do not closely associate with enzymes, as their polyester counterparts do, and the reactive radicals that are generated cannot chemically modify the macromolecular host. This study provides molecular guidance towards enzyme-polymer pairing and the selection of enzyme protectants to modulate substrate selectivity and optimize biocatalytic pathways. The results also highlight the need for in-depth research in solid-state enzymology, especially in multi-step enzymatic cascades, to tackle chemically dormant substrates without creating secondary environmental contamination and/or biosafety concerns.

The increasing relevance of cascade reactions in biocatalysis has sparked a great interest for enzyme co-immobilization. Enzyme co-immobilization allows access to some kinetic advantages that in some instances are necessary to get the desired product, avoiding side-reactions. However, the kinetic effect is very relevant mainly at the initial reaction rates, while it may be less relevant in the whole reaction course, depending on the kinetic parameters of the involved enzymes. This review not only critically discusses the advantages but also the drawbacks of enzymes co-immobilization: immobilization on the same support and surface, under similar conditions, discarding the whole biocatalyst when one of the co-immobilized enzymes is inactivated. We will discuss when co-immobilization is almost compulsory, when the advantages of co-immobilization may not be enough to compensate their problems and when it should be fully discarded. The co-immobilization of cofactors and enzymes bears special interest, as this can open up the opportunity to the building of artificial cells and extremely complex one-pot transformations. Finally, some recent strategies to overcome some the co-immobilization problems will be presented.

Successfully interfacing enzymes and biomachinery with polymers affords on-demand modification and/or programmable degradation during the manufacture, utilization and disposal of plastics, but requires controlled biocatalysis in solid matrices with macromolecular substrates1-7. Embedding enzyme microparticles speeds up polyester degradation, but compromises host properties and unintentionally accelerates the formation of microplastics with partial polymer degradation6,8,9. Here we show that by nanoscopically dispersing enzymes with deep active sites, semi-crystalline polyesters can be degraded primarily via chain-end-mediated processive depolymerization with programmable latency and material integrity, akin to polyadenylation-induced messenger RNA decay10. It is also feasible to achieve processivity with enzymes that have surface-exposed active sites by engineering enzyme-protectant-polymer complexes. Poly(caprolactone) and poly(lactic acid) containing less than 2 weight per cent enzymes are depolymerized in days, with up to 98 per cent polymer-to-small-molecule conversion in standard soil composts and household tap water, completely eliminating current needs to separate and landfill their products in compost facilities. Furthermore, oxidases embedded in polyolefins retain their activities. However, hydrocarbon polymers do not closely associate with enzymes, as their polyester counterparts do, and the reactive radicals that are generated cannot chemically modify the macromolecular host. This study provides molecular guidance towards enzyme-polymer pairing and the selection of enzyme protectants to modulate substrate selectivity and optimize biocatalytic pathways. The results also highlight the need for in-depth research in solid-state enzymology, especially in multi-step enzymatic cascades, to tackle chemically dormant substrates without creating secondary environmental contamination and/or biosafety concerns.

The reactivity of solid particles of two forms of TiO2, rutile and anatase, with H2O2 vapour was investigated for use as reactive coatings protecting granular formulations of enzymes. Reactivity tests using potassium titanium oxide oxalate as a competition probe showed a high reactivity of anatase at both high and low H2O2 pressure, whereas rutile was moderately reactive only at high H2O2 pressure. Heating H2O2-treated rutile and anatase led to release of oxygen, but not of surface-bound H2O2. For rutile, electron spin resonance measurements showed formation of radical intermediates. Anatase showed a mechanism comprising reversible binding of H2O2 and catalytic cleavage of H2O2 without formation of radicals. Anatase-coated detergent enzyme granules confirmed the protection against H2O2 vapour. Overall, this work shows the potential of anatase as a reactive coating material in laundry granules as an alternative to the current formulation.

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M-1s-1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M-1s-1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M-1s-1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M-1s-1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome in vitro We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.

The increasing relevance of cascade reactions in biocatalysis has sparked a great interest for enzyme co-immobilization. Enzyme co-immobilization allows access to some kinetic advantages that in some instances are necessary to get the desired product, avoiding side-reactions. However, the kinetic effect is very relevant mainly at the initial reaction rates, while it may be less relevant in the whole reaction course, depending on the kinetic parameters of the involved enzymes. This review not only critically discusses the advantages but also the drawbacks of enzymes co-immobilization: immobilization on the same support and surface, under similar conditions, discarding the whole biocatalyst when one of the co-immobilized enzymes is inactivated. We will discuss when co-immobilization is almost compulsory, when the advantages of co-immobilization may not be enough to compensate their problems and when it should be fully discarded. The co-immobilization of cofactors and enzymes bears special interest, as this can open up the opportunity to the building of artificial cells and extremely complex one-pot transformations. Finally, some recent strategies to overcome some the co-immobilization problems will be presented.

The enzymatic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxy-methylfurfural (HMF) has gained interest in recent years, as the renewable precursor of poly(ethylene-2,5-furandicarboxylate) (PEF). 5-Hydroxymethylfurfural oxidases (HMFOs) form a flavoenzyme family with genes annotated in a dozen bacterial species, but only one enzyme purified and characterized to date (after heterologous expression of a Methylovorus sp hmfo gene). This oxidase acts on both furfuryl alcohols and aldehydes being, therefore, able to catalyze the conversion of HMF into FDCA through 2,5-diformylfuran (DFF) and 2,5-formylfurancarboxylic acid (FFCA), with the only need of oxygen as cosubstrate. To enlarge the repertoire of HMFO enzymes available, genetic databases were screened for putative hmfo genes, followed by heterologous expression in Escherichia coli After unsuccessful trials with other bacterial hmfo genes, HMFOs from two Pseudomonas species were produced as active soluble enzymes, purified and characterized. The Methylovorus sp enzyme was also produced and purified in parallel for comparison. Enzyme stability against temperature, pH and hydrogen peroxide, three key aspects for application, were evaluated (together with optimal conditions for activity) revealing differences between the three HMFOs. Also the kinetic parameters for HMF, DFF and FFCA oxidation were determined, having the new HMFOs higher efficiencies for the oxidation of FFCA, which constitutes the bottleneck in the enzymatic route for FDCA production. These results were used to set up the best conditions for FDCA production by each enzyme, attaining a compromise between optimal activity and half-life under different operation conditions., IMPORTANCE: HMFO is the only enzyme described to date catalyzing by itself the three consecutive oxidation steps to produce FDCA from HMF. Unfortunately, only one HMFO enzyme is currently available for biotechnological application. This availability is enlarged here by the identification, heterologous production, purification and characterization of two new HMFOs from Pseudomonas nitroreducens and an unidentified Pseudomonas species. Compared to the previously known Methylovorus HMFO, the new enzyme from P. nitroreducens exhibits better performance for FDCA production in wider pH and temperature ranges, with higher tolerance to the hydrogen peroxide formed, longer half-life during oxidation, and higher yield and total turnover number in long-term conversions under optimized conditions. All these features are relevant properties for the industrial production of FDCA. In summary, gene screening and heterologous expression can facilitate the selection and improvement of HMFO enzymes as biocatalysts for the enzymatic synthesis of renewable building blocks in the production of bioplastics.

Biocatalysis is the branch of science at the intersection between chemistry and biology and specifcally dedicated to the application of natural evolvable catalysts, i.e. enzymes, in human-designed chemical processes. Among the array of promising biocatalysts, transaminases (EC 2.6.1.x) are possibly one of the enzyme classes with the largest unrealized potential. Fast inactivation, poor acceptance towards unnatural substrates and limited tolerance to cosolvents are some of the main factors hampering their implementation in chemical synthesis. In the present thesis work advances in both transaminase application and molecular understanding are presented. Indeed, these two topics are deeply interconnected, as a better molecular understanding is expected to ease the generation of novel enzyme variants suitable for new desired applications. From the application perspective, the design of an effective one-pot transaminase-based racemization system offers new possibilities for the design of fully biocatalytic dynamic kinetic resolutions of valuable chiral amines. Similarly, the successful structure-guided redesign of the small substrate binding pocket of the Chromobacterium violaceum (S)-selective transaminase (Cv-TA) granted access to a new enzyme variant active on semi-preparative scale towards the unnatural substrate 1,2-diphenylethylamine. From the molecular understanding perspective, the combination of crystallographic and computational techniques led to the formulation of a dimer dissociation model valid for Cv-TA and possibly for other enzymes belonging to the same fold type. This model, which aided the improvement of the Cv-TA stability by structure-based engineering, will hopefully enable similar results in other structurally related enzymes., Biokatalys är den del av naturvetenskapen mellan kemi och biologi som är specifkt inriktad på tillämpningar av naturligt utvecklade biokatalysatorer, dvs enzymer, i av människan skapade kemiska processer. Av alla lovande biokatalysatorer som existerar, är transaminaser (EC 2.6.1.x) kanske den enzymkategori som har störst outnyttjad potential. Snabb inaktivering, låg acceptans mot icke- naturliga substrat och en begränsad tolerans mot organiska lösningsmedel är några av de huvudsakliga faktorerna som begränsar användningen i kemiska synteser. I denna avhandling presenteras både framsteg inom transaminas-tillämpningar och molekylär förståelse. Dessa är i grunden djupt sammanhängande, då en bättre molekylär förståelse kan förväntas underlätta skapandet av tidigare okända enzymer som kan användas i nya önskade tillämpningar. Från ett applikationsperspektiv, skapar designen av ett effektivt en- kärls transaminasbaserat racemiserings-system nya möjligheter att designa biokatalytiska kinetiska resolveringar av värdefulla kirala aminer. På ett likartat sätt skapar den nya strukturbaserade designen av en liten substratbindande fcka i det (S)-selektiva transaminaset från Chromobacterium violaceum (Cv-TA) en ny enzymvariant som är aktiv i semi-preparativ skala med det icke-naturliga substratet 1,2- difenyletylamin. Från perspektivet av molekylär förståelse, ledde kombinationen av kristallograf och beräkningsteknik till formuleringen av en modell för dimer dissociationen av Cv-TA och som möjligtvis också gäller andra enzymer av samma struktur-typ. Med stöd av denna modell, har stabiliteten hos Cv-TA förbättrats med strukturbaserad modellering. Liknande resultat för strukturellt besläktade enzymer kommer förhoppningsvis produceras., La biocatalisi è quel ramo delle discipline scientifche situato all’intersezione tra chimica a biologia e dedicato all’applicazione di enzimi in processi chimici messi a punto dall’uomo. La classe enzimatica delle transaminasi (EC 2.6.1.x) è forse tra quelle con il più grande potenziale irrealizzato in questo settore. La rapida inattivazione, scarsa attività verso substrati non naturali e limitata stabilità in presenza di cosolventi ne limitano infatti l’uso in applicazioni di sintesi organica. Il lavoro presentato in questa tesi è dedicato all’avanzamento delle possibilità applicative delle transaminasi e al raggiungimento di una loro migliore comprensione a livello molecolare. Questi due aspetti sono sostanzialmente interconnessi, dal momento che una più profonda comprensione teorica può contribuire al successo di strategie di ingegnerizzatione dirette all’ottenimento di varianti enzimatiche adatte per particolari applicazioni. Nell’ambito applicativo, l’ideazione di un sistema basato sull’uso di transaminasi per la racemizzazione in one-pot di ammine chirali offre nuove possibilità per la risoluzione cinetica dinamica di questa classe di molecole. Allo stesso tempo, l’ingegnerizzazione della tasca minore del sito attivo della transaminasi (S)-selettiva isolata da Chromobacterium violaceum (Cv-TA) ne ha ampliato l’utilizzo per la risoluzione su scala semi-preparativa del substrato non-naturale 1,2- difeniletilamine. Nell’ambito dell’avanzamento teorico, la combinazione di tecniche cristallografche e computazionali ha portato alla formulazione di un modello in grado di descrivere il processo di dissociazione dell’organizzazione dimerica valido per Cv-TA e, verosimilmente, per altri enzimi appartenenti alla medesima classe strutturale. Questo modello ha guidato con successo l’ingegnerizzazione di Cv- TA per migliorarne la stabilità ed è possibile che possa contribuire al raggiungimento di risultati simili in altri enzimi strutturalmente correlati., QC 2019-10-01

Biocatalysis is the branch of science at the intersection between chemistry and biology and specifcally dedicated to the application of natural evolvable catalysts, i.e. enzymes, in human-designed chemical processes. Among the array of promising biocatalysts, transaminases (EC 2.6.1.x) are possibly one of the enzyme classes with the largest unrealized potential. Fast inactivation, poor acceptance towards unnatural substrates and limited tolerance to cosolvents are some of the main factors hampering their implementation in chemical synthesis. In the present thesis work advances in both transaminase application and molecular understanding are presented. Indeed, these two topics are deeply interconnected, as a better molecular understanding is expected to ease the generation of novel enzyme variants suitable for new desired applications. From the application perspective, the design of an effective one-pot transaminase-based racemization system offers new possibilities for the design of fully biocatalytic dynamic kinetic resolutions of valuable chiral amines. Similarly, the successful structure-guided redesign of the small substrate binding pocket of the Chromobacterium violaceum (S)-selective transaminase (Cv-TA) granted access to a new enzyme variant active on semi-preparative scale towards the unnatural substrate 1,2-diphenylethylamine. From the molecular understanding perspective, the combination of crystallographic and computational techniques led to the formulation of a dimer dissociation model valid for Cv-TA and possibly for other enzymes belonging to the same fold type. This model, which aided the improvement of the Cv-TA stability by structure-based engineering, will hopefully enable similar results in other structurally related enzymes., Biokatalys är den del av naturvetenskapen mellan kemi och biologi som är specifkt inriktad på tillämpningar av naturligt utvecklade biokatalysatorer, dvs enzymer, i av människan skapade kemiska processer. Av alla lovande biokatalysatorer som existerar, är transaminaser (EC 2.6.1.x) kanske den enzymkategori som har störst outnyttjad potential. Snabb inaktivering, låg acceptans mot icke- naturliga substrat och en begränsad tolerans mot organiska lösningsmedel är några av de huvudsakliga faktorerna som begränsar användningen i kemiska synteser. I denna avhandling presenteras både framsteg inom transaminas-tillämpningar och molekylär förståelse. Dessa är i grunden djupt sammanhängande, då en bättre molekylär förståelse kan förväntas underlätta skapandet av tidigare okända enzymer som kan användas i nya önskade tillämpningar. Från ett applikationsperspektiv, skapar designen av ett effektivt en- kärls transaminasbaserat racemiserings-system nya möjligheter att designa biokatalytiska kinetiska resolveringar av värdefulla kirala aminer. På ett likartat sätt skapar den nya strukturbaserade designen av en liten substratbindande fcka i det (S)-selektiva transaminaset från Chromobacterium violaceum (Cv-TA) en ny enzymvariant som är aktiv i semi-preparativ skala med det icke-naturliga substratet 1,2- difenyletylamin. Från perspektivet av molekylär förståelse, ledde kombinationen av kristallograf och beräkningsteknik till formuleringen av en modell för dimer dissociationen av Cv-TA och som möjligtvis också gäller andra enzymer av samma struktur-typ. Med stöd av denna modell, har stabiliteten hos Cv-TA förbättrats med strukturbaserad modellering. Liknande resultat för strukturellt besläktade enzymer kommer förhoppningsvis produceras., La biocatalisi è quel ramo delle discipline scientifche situato all’intersezione tra chimica a biologia e dedicato all’applicazione di enzimi in processi chimici messi a punto dall’uomo. La classe enzimatica delle transaminasi (EC 2.6.1.x) è forse tra quelle con il più grande potenziale irrealizzato in questo settore. La rapida inattivazione, scarsa attività verso substrati non naturali e limitata stabilità in presenza di cosolventi ne limitano infatti l’uso in applicazioni di sintesi organica. Il lavoro presentato in questa tesi è dedicato all’avanzamento delle possibilità applicative delle transaminasi e al raggiungimento di una loro migliore comprensione a livello molecolare. Questi due aspetti sono sostanzialmente interconnessi, dal momento che una più profonda comprensione teorica può contribuire al successo di strategie di ingegnerizzatione dirette all’ottenimento di varianti enzimatiche adatte per particolari applicazioni. Nell’ambito applicativo, l’ideazione di un sistema basato sull’uso di transaminasi per la racemizzazione in one-pot di ammine chirali offre nuove possibilità per la risoluzione cinetica dinamica di questa classe di molecole. Allo stesso tempo, l’ingegnerizzazione della tasca minore del sito attivo della transaminasi (S)-selettiva isolata da Chromobacterium violaceum (Cv-TA) ne ha ampliato l’utilizzo per la risoluzione su scala semi-preparativa del substrato non-naturale 1,2- difeniletilamine. Nell’ambito dell’avanzamento teorico, la combinazione di tecniche cristallografche e computazionali ha portato alla formulazione di un modello in grado di descrivere il processo di dissociazione dell’organizzazione dimerica valido per Cv-TA e, verosimilmente, per altri enzimi appartenenti alla medesima classe strutturale. Questo modello ha guidato con successo l’ingegnerizzazione di Cv- TA per migliorarne la stabilità ed è possibile che possa contribuire al raggiungimento di risultati simili in altri enzimi strutturalmente correlati., QC 2019-10-01

Sulfotransferase 1A1 (SULT1A1) is responsible for catalyzing various types of endogenous and exogenous compounds. Accumulating data indicates that the polymorphism rs9282861 (R213H) is responsible for inefficient enzymatic activity and associated with cancer progression. To characterize the detailed functional consequences of this mutation behind the loss-of-function of SULT1A1, the present study deployed molecular dynamics simulation to get insights into changes in the conformation and binding energy. The dynamics scenario of SULT1A1 in both wild and mutated types as well as with and without ligand showed that R213H induced local conformational changes, especially in the substrate-binding loop rather than impairing overall stability of the protein structure. The higher conformational changes were observed in the loop3 (residues, 235–263), turning loop conformation to A-helix and B-bridge, which ultimately disrupted the plasticity of the active site. This alteration reduced the binding site volume and hydrophobicity to decrease the binding affinity of the enzyme to substrates, which was highlighted by the MM-PBSA binding energy analysis. These findings highlight the key insights of structural consequences caused by R213H mutation, which would enrich the understanding regarding the role of SULT1A1 mutation in cancer development and also xenobiotics management to individuals in the different treatment stages.

Placental growth factor (PlGF), total soluble fms-like tyrosine-kinase 1 (sFlt-1) and its placental-specific variant, sFlt-1 e15a, show promise as biomarkers for the prediction and diagnosis of preeclampsia. This study describes the degradation of PlGF, sFlt-1 and sFlt-1 e15a within maternal serum and plasma to assist clinical implementation. Whole blood was refrigerated at 4 degreeC for up to 48 h prior to centrifugation for isolation of plasma and serum. PlGF and sFlt-1 were quantified using the B.R.A.H.M.S Kryptor Compact PLUS; sFlt-1 e15a via a custom ELISA. All three analytes are stable for at least 48 h at 4 degreeC. Serum and plasma performed comparably.Copyright © 2019 Elsevier Ltd

The addition of feed enzymes to livestock diets has contributed to significant increases in productivity over recent decades. The use of enzymes has been the most common in systems where enzyme delivery and diets can be easily managed, such as for poultry and pigs. Lick blocks supplement the forage diets of ruminants with nitrogen and minerals but not enzymes, due in part to concerns that block manufacturing temperatures would lead to unacceptable levels of enzyme degradation. The nutritional value of low quality pasture could be improved using enzyme supplemented lick blocks if enzymes remain active at the high lick block manufacturing temperatures. The aim of this study was to determine the extent of xylanase and phytase activity survival when exposed to the production of hot poured lick blocks. Lick block formulations and methods of manufacturing vary, so two enzyme containing molasses-based lick blocks were produced, one at 60°C and another at 100°C. The results showed that both the xylanase and phytase enzymes have high levels of survival at 60°C. In the 100°C lick block, the phytase displayed a half-life of ~10 min, whereas the xylanase retained 90% of the original activity after 30 min of exposure. The inherent thermostability of the enzymes were critical factors for enzyme survival and the enzymes were more stable in the lick blocks than in solution. The results indicate that it should be possible to add enzymes to lick blocks manufactured at elevated temperatures to enhance low quality pasture and thereby aid ruminant digestion and production.

One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. Our still limited understanding of the inactivation processes undermining the stability of wild-type transaminases represents an obstacle to improving their stability through enzyme engineering. In this paper we present a model describing the inactivation process of the well-characterized (S)-selective amine transaminase from Chromobacterium violaceum. The cornerstone of the model, supported by structural, computational, mutagenesis and biophysical data, is the central role of the catalytic lysine as a conformational switch. Upon breakage of the lysine-PLP Schiff base, the strain associated with the catalytically-active lysine conformation is dissipated in a slow relaxation process capable of triggering the known structural rearrangements occurring in the holo-to-apo transition and ultimately promoting dimer dissociation. Due to the occurrence in the literature of similar PLP-dependent inactivation models valid for other non-transaminase enzymes belonging to the same fold-class, the role of the catalytic lysine as conformational switch might extend beyond the transaminase enzyme group and offer new insight to drive future non-trivial engineering strategies., QC 20191011Non-duplicate with diva 1356294

In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results of experiments using different bioluminescent reporters, such as Akaluc and Antareas2, have been reported. Herein, we re-examined the inconsistency in several experimental settings and identified the factors, such as ATP dependency, stability in serum, and molecular sizes of luciferases, that contributed to the observed differences. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.

Cytotoxic protein aggregation-induced impairment of cell function and homeostasis are hallmarks of age-related neurodegenerative pathologies. As proteasomal degradation represents the major clearance pathway for oxidatively damaged proteins, a detailed understanding of the molecular events underlying its stress response is critical for developing strategies to maintain cell viability and function. Although the 26S proteasome has been shown to disassemble during oxidative stress, its conformational dynamics remains unclear. To this end, we have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress. This strategy comprises SILAC-based metabolic labeling, HB tag-based affinity purification, a 2-step cross-linking reaction consisting of mild in vivo formaldehyde and on-bead DSSO cross-linking, and multi-stage tandem mass spectrometry (MSn) to identify and quantify cross-links. This integrated workflow has been successfully applied to explore the molecular events underlying oxidative stress-dependent proteasomal regulation by comparative analyses of proteasome complex topologies from treated and untreated cells. Our results show that H2O2 treatment weakens the 19S-20S interaction within the 26S proteasome, along with reorganizations within the 19S and 20S subcomplexes. Altogether, this work sheds light on the mechanistic response of the 26S to acute oxidative stress, suggesting an intermediate proteasomal state(s) before H2O2-mediated dissociation of the 26S. The QXL-MS strategy presented here can be applied to study conformational changes of other protein complexes under different physiological conditions.

Much is known about the structure, function, and stability of the SecA motor ATPase that powers the secretion of periplasmic proteins across the inner membrane of Escherichia coli. Most studies of SecA are carried out in buffered sodium or potassium chloride salt solutions. However, the principal intracellular salt of E. coli is potassium glutamate (KGlu), which is known to stabilize folded proteins and protein-nucleic acid complexes. Here we report that KGlu stabilizes SecA, including its dimeric state, and increases its ATPase activity, suggesting that SecA is likely fully folded, stable, and active in vivo at 37°C. Furthermore, KGlu also stabilizes a precursor form of the secreted maltose-binding protein.

Biocatalysis is the branch of science at the intersection between chemistry and biology and specifcally dedicated to the application of natural evolvable catalysts, i.e. enzymes, in human-designed chemical processes. Among the array of promising biocatalysts, transaminases (EC 2.6.1.x) are possibly one of the enzyme classes with the largest unrealized potential. Fast inactivation, poor acceptance towards unnatural substrates and limited tolerance to cosolvents are some of the main factors hampering their implementation in chemical synthesis. In the present thesis work advances in both transaminase application and molecular understanding are presented. Indeed, these two topics are deeply interconnected, as a better molecular understanding is expected to ease the generation of novel enzyme variants suitable for new desired applications. From the application perspective, the design of an effective one-pot transaminase-based racemization system offers new possibilities for the design of fully biocatalytic dynamic kinetic resolutions of valuable chiral amines. Similarly, the successful structure-guided redesign of the small substrate binding pocket of the Chromobacterium violaceum (S)-selective transaminase (Cv-TA) granted access to a new enzyme variant active on semi-preparative scale towards the unnatural substrate 1,2-diphenylethylamine. From the molecular understanding perspective, the combination of crystallographic and computational techniques led to the formulation of a dimer dissociation model valid for Cv-TA and possibly for other enzymes belonging to the same fold type. This model, which aided the improvement of the Cv-TA stability by structure-based engineering, will hopefully enable similar results in other structurally related enzymes., Biokatalys är den del av naturvetenskapen mellan kemi och biologi som är specifkt inriktad på tillämpningar av naturligt utvecklade biokatalysatorer, dvs enzymer, i av människan skapade kemiska processer. Av alla lovande biokatalysatorer som existerar, är transaminaser (EC 2.6.1.x) kanske den enzymkategori som har störst outnyttjad potential. Snabb inaktivering, låg acceptans mot icke- naturliga substrat och en begränsad tolerans mot organiska lösningsmedel är några av de huvudsakliga faktorerna som begränsar användningen i kemiska synteser. I denna avhandling presenteras både framsteg inom transaminas-tillämpningar och molekylär förståelse. Dessa är i grunden djupt sammanhängande, då en bättre molekylär förståelse kan förväntas underlätta skapandet av tidigare okända enzymer som kan användas i nya önskade tillämpningar. Från ett applikationsperspektiv, skapar designen av ett effektivt en- kärls transaminasbaserat racemiserings-system nya möjligheter att designa biokatalytiska kinetiska resolveringar av värdefulla kirala aminer. På ett likartat sätt skapar den nya strukturbaserade designen av en liten substratbindande fcka i det (S)-selektiva transaminaset från Chromobacterium violaceum (Cv-TA) en ny enzymvariant som är aktiv i semi-preparativ skala med det icke-naturliga substratet 1,2- difenyletylamin. Från perspektivet av molekylär förståelse, ledde kombinationen av kristallograf och beräkningsteknik till formuleringen av en modell för dimer dissociationen av Cv-TA och som möjligtvis också gäller andra enzymer av samma struktur-typ. Med stöd av denna modell, har stabiliteten hos Cv-TA förbättrats med strukturbaserad modellering. Liknande resultat för strukturellt besläktade enzymer kommer förhoppningsvis produceras., La biocatalisi è quel ramo delle discipline scientifche situato all’intersezione tra chimica a biologia e dedicato all’applicazione di enzimi in processi chimici messi a punto dall’uomo. La classe enzimatica delle transaminasi (EC 2.6.1.x) è forse tra quelle con il più grande potenziale irrealizzato in questo settore. La rapida inattivazione, scarsa attività verso substrati non naturali e limitata stabilità in presenza di cosolventi ne limitano infatti l’uso in applicazioni di sintesi organica. Il lavoro presentato in questa tesi è dedicato all’avanzamento delle possibilità applicative delle transaminasi e al raggiungimento di una loro migliore comprensione a livello molecolare. Questi due aspetti sono sostanzialmente interconnessi, dal momento che una più profonda comprensione teorica può contribuire al successo di strategie di ingegnerizzatione dirette all’ottenimento di varianti enzimatiche adatte per particolari applicazioni. Nell’ambito applicativo, l’ideazione di un sistema basato sull’uso di transaminasi per la racemizzazione in one-pot di ammine chirali offre nuove possibilità per la risoluzione cinetica dinamica di questa classe di molecole. Allo stesso tempo, l’ingegnerizzazione della tasca minore del sito attivo della transaminasi (S)-selettiva isolata da Chromobacterium violaceum (Cv-TA) ne ha ampliato l’utilizzo per la risoluzione su scala semi-preparativa del substrato non-naturale 1,2- difeniletilamine. Nell’ambito dell’avanzamento teorico, la combinazione di tecniche cristallografche e computazionali ha portato alla formulazione di un modello in grado di descrivere il processo di dissociazione dell’organizzazione dimerica valido per Cv-TA e, verosimilmente, per altri enzimi appartenenti alla medesima classe strutturale. Questo modello ha guidato con successo l’ingegnerizzazione di Cv- TA per migliorarne la stabilità ed è possibile che possa contribuire al raggiungimento di risultati simili in altri enzimi strutturalmente correlati., QC 2019-10-01

Biocatalysis is the branch of science at the intersection between chemistry and biology and specifcally dedicated to the application of natural evolvable catalysts, i.e. enzymes, in human-designed chemical processes. Among the array of promising biocatalysts, transaminases (EC 2.6.1.x) are possibly one of the enzyme classes with the largest unrealized potential. Fast inactivation, poor acceptance towards unnatural substrates and limited tolerance to cosolvents are some of the main factors hampering their implementation in chemical synthesis. In the present thesis work advances in both transaminase application and molecular understanding are presented. Indeed, these two topics are deeply interconnected, as a better molecular understanding is expected to ease the generation of novel enzyme variants suitable for new desired applications. From the application perspective, the design of an effective one-pot transaminase-based racemization system offers new possibilities for the design of fully biocatalytic dynamic kinetic resolutions of valuable chiral amines. Similarly, the successful structure-guided redesign of the small substrate binding pocket of the Chromobacterium violaceum (S)-selective transaminase (Cv-TA) granted access to a new enzyme variant active on semi-preparative scale towards the unnatural substrate 1,2-diphenylethylamine. From the molecular understanding perspective, the combination of crystallographic and computational techniques led to the formulation of a dimer dissociation model valid for Cv-TA and possibly for other enzymes belonging to the same fold type. This model, which aided the improvement of the Cv-TA stability by structure-based engineering, will hopefully enable similar results in other structurally related enzymes., Biokatalys är den del av naturvetenskapen mellan kemi och biologi som är specifkt inriktad på tillämpningar av naturligt utvecklade biokatalysatorer, dvs enzymer, i av människan skapade kemiska processer. Av alla lovande biokatalysatorer som existerar, är transaminaser (EC 2.6.1.x) kanske den enzymkategori som har störst outnyttjad potential. Snabb inaktivering, låg acceptans mot icke- naturliga substrat och en begränsad tolerans mot organiska lösningsmedel är några av de huvudsakliga faktorerna som begränsar användningen i kemiska synteser. I denna avhandling presenteras både framsteg inom transaminas-tillämpningar och molekylär förståelse. Dessa är i grunden djupt sammanhängande, då en bättre molekylär förståelse kan förväntas underlätta skapandet av tidigare okända enzymer som kan användas i nya önskade tillämpningar. Från ett applikationsperspektiv, skapar designen av ett effektivt en- kärls transaminasbaserat racemiserings-system nya möjligheter att designa biokatalytiska kinetiska resolveringar av värdefulla kirala aminer. På ett likartat sätt skapar den nya strukturbaserade designen av en liten substratbindande fcka i det (S)-selektiva transaminaset från Chromobacterium violaceum (Cv-TA) en ny enzymvariant som är aktiv i semi-preparativ skala med det icke-naturliga substratet 1,2- difenyletylamin. Från perspektivet av molekylär förståelse, ledde kombinationen av kristallograf och beräkningsteknik till formuleringen av en modell för dimer dissociationen av Cv-TA och som möjligtvis också gäller andra enzymer av samma struktur-typ. Med stöd av denna modell, har stabiliteten hos Cv-TA förbättrats med strukturbaserad modellering. Liknande resultat för strukturellt besläktade enzymer kommer förhoppningsvis produceras., La biocatalisi è quel ramo delle discipline scientifche situato all’intersezione tra chimica a biologia e dedicato all’applicazione di enzimi in processi chimici messi a punto dall’uomo. La classe enzimatica delle transaminasi (EC 2.6.1.x) è forse tra quelle con il più grande potenziale irrealizzato in questo settore. La rapida inattivazione, scarsa attività verso substrati non naturali e limitata stabilità in presenza di cosolventi ne limitano infatti l’uso in applicazioni di sintesi organica. Il lavoro presentato in questa tesi è dedicato all’avanzamento delle possibilità applicative delle transaminasi e al raggiungimento di una loro migliore comprensione a livello molecolare. Questi due aspetti sono sostanzialmente interconnessi, dal momento che una più profonda comprensione teorica può contribuire al successo di strategie di ingegnerizzatione dirette all’ottenimento di varianti enzimatiche adatte per particolari applicazioni. Nell’ambito applicativo, l’ideazione di un sistema basato sull’uso di transaminasi per la racemizzazione in one-pot di ammine chirali offre nuove possibilità per la risoluzione cinetica dinamica di questa classe di molecole. Allo stesso tempo, l’ingegnerizzazione della tasca minore del sito attivo della transaminasi (S)-selettiva isolata da Chromobacterium violaceum (Cv-TA) ne ha ampliato l’utilizzo per la risoluzione su scala semi-preparativa del substrato non-naturale 1,2- difeniletilamine. Nell’ambito dell’avanzamento teorico, la combinazione di tecniche cristallografche e computazionali ha portato alla formulazione di un modello in grado di descrivere il processo di dissociazione dell’organizzazione dimerica valido per Cv-TA e, verosimilmente, per altri enzimi appartenenti alla medesima classe strutturale. Questo modello ha guidato con successo l’ingegnerizzazione di Cv- TA per migliorarne la stabilità ed è possibile che possa contribuire al raggiungimento di risultati simili in altri enzimi strutturalmente correlati., QC 2019-10-01

Biocatalysis is the branch of science at the intersection between chemistry and biology and specifcally dedicated to the application of natural evolvable catalysts, i.e. enzymes, in human-designed chemical processes. Among the array of promising biocatalysts, transaminases (EC 2.6.1.x) are possibly one of the enzyme classes with the largest unrealized potential. Fast inactivation, poor acceptance towards unnatural substrates and limited tolerance to cosolvents are some of the main factors hampering their implementation in chemical synthesis. In the present thesis work advances in both transaminase application and molecular understanding are presented. Indeed, these two topics are deeply interconnected, as a better molecular understanding is expected to ease the generation of novel enzyme variants suitable for new desired applications. From the application perspective, the design of an effective one-pot transaminase-based racemization system offers new possibilities for the design of fully biocatalytic dynamic kinetic resolutions of valuable chiral amines. Similarly, the successful structure-guided redesign of the small substrate binding pocket of the Chromobacterium violaceum (S)-selective transaminase (Cv-TA) granted access to a new enzyme variant active on semi-preparative scale towards the unnatural substrate 1,2-diphenylethylamine. From the molecular understanding perspective, the combination of crystallographic and computational techniques led to the formulation of a dimer dissociation model valid for Cv-TA and possibly for other enzymes belonging to the same fold type. This model, which aided the improvement of the Cv-TA stability by structure-based engineering, will hopefully enable similar results in other structurally related enzymes., Biokatalys är den del av naturvetenskapen mellan kemi och biologi som är specifkt inriktad på tillämpningar av naturligt utvecklade biokatalysatorer, dvs enzymer, i av människan skapade kemiska processer. Av alla lovande biokatalysatorer som existerar, är transaminaser (EC 2.6.1.x) kanske den enzymkategori som har störst outnyttjad potential. Snabb inaktivering, låg acceptans mot icke- naturliga substrat och en begränsad tolerans mot organiska lösningsmedel är några av de huvudsakliga faktorerna som begränsar användningen i kemiska synteser. I denna avhandling presenteras både framsteg inom transaminas-tillämpningar och molekylär förståelse. Dessa är i grunden djupt sammanhängande, då en bättre molekylär förståelse kan förväntas underlätta skapandet av tidigare okända enzymer som kan användas i nya önskade tillämpningar. Från ett applikationsperspektiv, skapar designen av ett effektivt en- kärls transaminasbaserat racemiserings-system nya möjligheter att designa biokatalytiska kinetiska resolveringar av värdefulla kirala aminer. På ett likartat sätt skapar den nya strukturbaserade designen av en liten substratbindande fcka i det (S)-selektiva transaminaset från Chromobacterium violaceum (Cv-TA) en ny enzymvariant som är aktiv i semi-preparativ skala med det icke-naturliga substratet 1,2- difenyletylamin. Från perspektivet av molekylär förståelse, ledde kombinationen av kristallograf och beräkningsteknik till formuleringen av en modell för dimer dissociationen av Cv-TA och som möjligtvis också gäller andra enzymer av samma struktur-typ. Med stöd av denna modell, har stabiliteten hos Cv-TA förbättrats med strukturbaserad modellering. Liknande resultat för strukturellt besläktade enzymer kommer förhoppningsvis produceras., La biocatalisi è quel ramo delle discipline scientifche situato all’intersezione tra chimica a biologia e dedicato all’applicazione di enzimi in processi chimici messi a punto dall’uomo. La classe enzimatica delle transaminasi (EC 2.6.1.x) è forse tra quelle con il più grande potenziale irrealizzato in questo settore. La rapida inattivazione, scarsa attività verso substrati non naturali e limitata stabilità in presenza di cosolventi ne limitano infatti l’uso in applicazioni di sintesi organica. Il lavoro presentato in questa tesi è dedicato all’avanzamento delle possibilità applicative delle transaminasi e al raggiungimento di una loro migliore comprensione a livello molecolare. Questi due aspetti sono sostanzialmente interconnessi, dal momento che una più profonda comprensione teorica può contribuire al successo di strategie di ingegnerizzatione dirette all’ottenimento di varianti enzimatiche adatte per particolari applicazioni. Nell’ambito applicativo, l’ideazione di un sistema basato sull’uso di transaminasi per la racemizzazione in one-pot di ammine chirali offre nuove possibilità per la risoluzione cinetica dinamica di questa classe di molecole. Allo stesso tempo, l’ingegnerizzazione della tasca minore del sito attivo della transaminasi (S)-selettiva isolata da Chromobacterium violaceum (Cv-TA) ne ha ampliato l’utilizzo per la risoluzione su scala semi-preparativa del substrato non-naturale 1,2- difeniletilamine. Nell’ambito dell’avanzamento teorico, la combinazione di tecniche cristallografche e computazionali ha portato alla formulazione di un modello in grado di descrivere il processo di dissociazione dell’organizzazione dimerica valido per Cv-TA e, verosimilmente, per altri enzimi appartenenti alla medesima classe strutturale. Questo modello ha guidato con successo l’ingegnerizzazione di Cv- TA per migliorarne la stabilità ed è possibile che possa contribuire al raggiungimento di risultati simili in altri enzimi strutturalmente correlati., QC 2019-10-01