Enzymatic Hydrolysis of Human Milk Oligosaccharides. The Molecular Mechanism of Bifidobacterium Bifidum Lacto-N-biosidase

Bifidobacterium bifidum lacto-N-biosidase (LnbB) is a critical enzyme for the degradation of human milk oligosaccharides in the gut microbiota of breast-fed infants. Guided by recent crystal structures, we unveil its molecular mechanism of catalysis using QM/MM metadynamics. We show that the oligosaccharide substrate follows 1S3/1,4B → [4E]¿ → 4C1/4H5 and 4C1/4H5 → [4E/4H5]¿ → 1,4B conformational itineraries for the two successive reaction steps, with reaction free energy barriers in agreement with experiments. The simulations also identify a critical histidine (His263) that switches between two orientations to modulate the pKa of the acid/base residue, facilitating catalysis. The reaction intermediate of LnbB is best depicted as an oxazolinium ion, with a minor population of neutral oxazoline. The present study sheds light on the processing of oligosaccharides of the early life microbiota and will be useful for the engineering of LnbB and similar glycosidases for biocatalysis

Ratiometric Nanothermometer Based on a Radical Excimer for In Vivo Sensing

Ratiometric fluorescent nanothermometers with near-infrared emission play an important role in in vivo sensing since they can be used as intracellular thermal sensing probes with high spatial resolution and high sensitivity, to investigate cellular functions of interest in diagnosis and therapy, where current approaches are not effective. Herein, the temperature-dependent fluorescence of organic nanoparticles is designed, synthesized, and studied based on the dual emission, generated by monomer and excimer species, of the tris(2,4,6-trichlorophenyl)methyl radical (TTM) doping organic nanoparticles (TTMd-ONPs), made of optically neutral tris(2,4,6-trichlorophenyl)methane (TTM-αH), acting as a matrix. The excimer emission intensity of TTMd-ONPs decreases with increasing temperatures whereas the monomer emission is almost independent and can be used as an internal reference. TTMd-ONPs show a great temperature sensitivity (3.4% K-1 at 328 K) and a wide temperature response at ambient conditions with excellent reversibility and high colloidal stability. In addition, TTMd-ONPs are not cytotoxic and their ratiometric outputs are unaffected by changes in the environment. Individual TTMd-ONPs are able to sense temperature changes at the nano-microscale. In vivo thermometry experiments in Caenorhabditis elegans (C. elegans) worms show that TTMd-ONPs can locally monitor internal body temperature changes with spatio-temporal resolution and high sensitivity, offering multiple applications in the biological nanothermometry field.© 2023 The Authors. Small published by Wiley-VCH GmbH

Computational insights into carbohydrate epimerase mechanisms

Programa de Doctorat en Química Orgànica[eng] Carbohydrates, as the most abundant biomolecules, play a myriad of roles and functions in biological systems. Unlike the building blocks of proteins, amino acids, whose chemical structure can vary significantly, monosaccharides, the building blocks of carbohydrates, can exhibit small differences in their chemical structure that lead to a very different chemistry. A defining characteristic of monosaccharides is their stereochemistry. Just four or five stereogenic centres in each monosaccharide leads to a vast array of structural possibilities. Given the subtle structural differences between monosaccharides, the enzymes that modify carbohydrates (carbohydrate-active enzymes, CAZymes) must exhibit precise specificity for their substrates. Computational approaches have proven of great value in elucidating the enzymatic mechanisms of CAZymes, particularly in determining the conformation of monosaccharides within the active site during catalysis. Carbohydrate epimerases, a subset of CAZymes, modify the stereogenic centres of carbohydrates. Despite their key roles in biological organisms, such as catalysing the interconversion between glucose and galactose, they remain relatively understudied. The inherent challenge in studying epimerases lies in their need for precise control over the conformation and positioning of the carbohydrate within the active site, given that epimerisation is an equilibrium reaction (i.e., reactant and product have similar energy) and the reactant, intermediates and product are structurally similar. The main applications of carbohydrate epimerases are in biomedicine and biotechnology. Their important roles in humans and other organisms make the research of this group of enzymes essential for biomedical purposes, such as the design of antibiotics. They are also therapeutic targets for the treatment of diseases, such as galactosemia. Furthermore, carbohydrate epimerases can be used to synthesize rare sugars from common ones. While our research group has extensive expertise in CAZymes, it had no experience in carbohydrate epimerases before starting this Thesis. This Thesis summarizes our recent work to uncover some of the innumerable intricacies of carbohydrate epimerases. Following a general introduction and a chapter on the used methods, we elucidate the details of human GDP-L-fucose synthase mechanism. In subsequent chapters, we elucidate the complete mechanism of another biomedically relevant epimerase, UDP-D-glucuronic acid 4- epimerase. - Chapter I: Introduction In this chapter, we introduce the main features of carbohydrates, such as their stereochemistry and conformation, followed by a description of carbohydrate epimerases classification and their mechanisms. The main objectives of this Thesis are detailed at the end of the chapter. - Chapter II: Methods We provide a clear overview of all the computational methodologies employed throughout this Thesis. - Chapter III: Molecular mechanism of regio-selective catalysis in human GDP-L- fucose synthase We uncover the full conformational itinerary of the sugar within the active site human GDP-L-fucose synthase (GFS) throughout its whole catalytic process, and we also elucidate the strategy that the enzyme uses to avoid the premature reduction of the sugar through the precise control of sugar conformation and positioning within the active site. - Chapter IV: Sugar oxidation and rotation in UDP-glucuronic acid C4-epimerisation process We study the oxidation mechanism of the substrate and the elusive mechanism for the rotation of the 4-keto-intermediate within the enzyme active site in Bacillus cereus UDP- D-glucuronic acid C4-epimerase (UGAE). We also discussed how mutations of an important active site residue (Arg185 ) affect catalysis. - Chapter V: Sugar reduction and proton shuttle in UDP-D-glucuronic acid C4- epimerisation process We investigate the mechanism of reduction of UDP-4-keto-hexuronic acid intermediate to UDP-galacturonic acid, which completes the full catalytic mechanism of UDP-D- glucuronic acid C4-epimerase (UGAE).[cat] Els carbohidrats son les biomolècules més abundants i tenen rols i funcions molt importants en els sistemes biològics. Els aminoàcids, que son els blocs fonamentals de les proteïnes, tenen estructures molt diferents entre ells, no obstant, els monosacàrids, que constitueixen els carbohidrats, tenen estructures químiques molt similars. Un de les característiques definitòries dels monosacàrids és la estereoquímica, doncs habitualment cada monosacàrid te uns quatre o cinc centres estereogènics, i això dona obra un gran ventall de possibilitats. Els enzims que es dediquen a invertir la seva estereoquímica són les epimerases de carbohidrats. Aquests enzims necessiten tenir un control molt específic dels substrats, ja que l’estructura del reactiu, dels intermedis i del producte són molt similars, i han de poder controlar bé com el sucre està col·locat en el centre actiu per poder catalitzar la reacció correcte. Conèixer bé els seus mecanismes catalítics és important a nivell mèdic per desenvolupar tractaments i diagnòstics per malalties relacionades amb aquests enzims. A més, també són importants a nivell biotecnològic per sintetitzar carbohidrats o derivats poc comuns a partir dels que carbohidrats més abundants. Donat les importants aplicacions i els escassos estudis d’aquest grup d’enzims, en aquesta tesi s’exploren els detalls mecanístics de dues epimerases a través de mètodes computacionals complementats amb resultats experimentals dels nostres col·laboradors. Les epimerases de carbohidrats estudiades són la sintasa de GDP-L-fucosa i la 4-epimerasa d’UDP-àcid glucurònic

Computational insights into carbohydrate epimerase mechanisms

[eng] Carbohydrates, as the most abundant biomolecules, play a myriad of roles and functions in biological systems. Unlike the building blocks of proteins, amino acids, whose chemical structure can vary significantly, monosaccharides, the building blocks of carbohydrates, can exhibit small differences in their chemical structure that lead to a very different chemistry. A defining characteristic of monosaccharides is their stereochemistry. Just four or five stereogenic centres in each monosaccharide leads to a vast array of structural possibilities. Given the subtle structural differences between monosaccharides, the enzymes that modify carbohydrates (carbohydrate-active enzymes, CAZymes) must exhibit precise specificity for their substrates. Computational approaches have proven of great value in elucidating the enzymatic mechanisms of CAZymes, particularly in determining the conformation of monosaccharides within the active site during catalysis. Carbohydrate epimerases, a subset of CAZymes, modify the stereogenic centres of carbohydrates. Despite their key roles in biological organisms, such as catalysing the interconversion between glucose and galactose, they remain relatively understudied. The inherent challenge in studying epimerases lies in their need for precise control over the conformation and positioning of the carbohydrate within the active site, given that epimerisation is an equilibrium reaction (i.e., reactant and product have similar energy) and the reactant, intermediates and product are structurally similar. The main applications of carbohydrate epimerases are in biomedicine and biotechnology. Their important roles in humans and other organisms make the research of this group of enzymes essential for biomedical purposes, such as the design of antibiotics. They are also therapeutic targets for the treatment of diseases, such as galactosemia. Furthermore, carbohydrate epimerases can be used to synthesize rare sugars from common ones. While our research group has extensive expertise in CAZymes, it had no experience in carbohydrate epimerases before starting this Thesis. This Thesis summarizes our recent work to uncover some of the innumerable intricacies of carbohydrate epimerases. Following a general introduction and a chapter on the used methods, we elucidate the details of human GDP-L-fucose synthase mechanism. In subsequent chapters, we elucidate the complete mechanism of another biomedically relevant epimerase, UDP-D-glucuronic acid 4- epimerase. - Chapter I: Introduction In this chapter, we introduce the main features of carbohydrates, such as their stereochemistry and conformation, followed by a description of carbohydrate epimerases classification and their mechanisms. The main objectives of this Thesis are detailed at the end of the chapter. - Chapter II: Methods We provide a clear overview of all the computational methodologies employed throughout this Thesis. - Chapter III: Molecular mechanism of regio-selective catalysis in human GDP-L- fucose synthase We uncover the full conformational itinerary of the sugar within the active site human GDP-L-fucose synthase (GFS) throughout its whole catalytic process, and we also elucidate the strategy that the enzyme uses to avoid the premature reduction of the sugar through the precise control of sugar conformation and positioning within the active site. - Chapter IV: Sugar oxidation and rotation in UDP-glucuronic acid C4-epimerisation process We study the oxidation mechanism of the substrate and the elusive mechanism for the rotation of the 4-keto-intermediate within the enzyme active site in Bacillus cereus UDP- D-glucuronic acid C4-epimerase (UGAE). We also discussed how mutations of an important active site residue (Arg185 ) affect catalysis. - Chapter V: Sugar reduction and proton shuttle in UDP-D-glucuronic acid C4- epimerisation process We investigate the mechanism of reduction of UDP-4-keto-hexuronic acid intermediate to UDP-galacturonic acid, which completes the full catalytic mechanism of UDP-D- glucuronic acid C4-epimerase (UGAE).[cat] Els carbohidrats son les biomolècules més abundants i tenen rols i funcions molt importants en els sistemes biològics. Els aminoàcids, que son els blocs fonamentals de les proteïnes, tenen estructures molt diferents entre ells, no obstant, els monosacàrids, que constitueixen els carbohidrats, tenen estructures químiques molt similars. Un de les característiques definitòries dels monosacàrids és la estereoquímica, doncs habitualment cada monosacàrid te uns quatre o cinc centres estereogènics, i això dona obra un gran ventall de possibilitats. Els enzims que es dediquen a invertir la seva estereoquímica són les epimerases de carbohidrats. Aquests enzims necessiten tenir un control molt específic dels substrats, ja que l’estructura del reactiu, dels intermedis i del producte són molt similars, i han de poder controlar bé com el sucre està col·locat en el centre actiu per poder catalitzar la reacció correcte. Conèixer bé els seus mecanismes catalítics és important a nivell mèdic per desenvolupar tractaments i diagnòstics per malalties relacionades amb aquests enzims. A més, també són importants a nivell biotecnològic per sintetitzar carbohidrats o derivats poc comuns a partir dels que carbohidrats més abundants. Donat les importants aplicacions i els escassos estudis d’aquest grup d’enzims, en aquesta tesi s’exploren els detalls mecanístics de dues epimerases a través de mètodes computacionals complementats amb resultats experimentals dels nostres col·laboradors. Les epimerases de carbohidrats estudiades són la sintasa de GDP-L-fucosa i la 4-epimerasa d’UDP-àcid glucurònic