Computational insights into carbohydrate epimerase mechanisms

Abstract

[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.