Simulations of glycoside hydrolase and phosphorylase reaction mechanism: families GH20, GH29, GH129 and GH130

Abstract

[eng] The most abundant family of biomolecules on earth are carbohydrates. Also known as glycans, sugars or saccharides, are based on polyhydroxycarbonyl backbones with a vast array of chemical and structural modifications, which confer them different physico-chemical properties. They participate in many biological processes related to structural, energy storage and molecular signaling functions, and the many possibilities regarding structure and function of carbohydrates open questions in the field of glycobiology. The simplest form of carbohydrates, monosaccharides, assemble through glycosidic bonds to form larger structures (oligo- or polysaccharides) or glycoconjugates in combination with other families of biomolecules (like glycoproteins). Carbohydrates are flexible: from the atomic positions of the atoms forming a monosaccharide ring, to the rotation of the linkages between sugar units, it is a property that confers them the possibility to arrange their atoms in different positions resulting in different conformations, a key aspect in processes where they take part in. Carbohydrates undergo chemical changes in the form of degradation, synthesis and modifications in the several biological processes where they participate. Operating these reactions, we find the Carbohydrate Active Enzymes (CAZYmes), whose functions are increasingly being discovered. The study of CAZYmes has attracted research attention, as it opens windows in how complex metabolic pathways protagonized by carbohydrates work and paves new paths for medical, biotechnological and environmental applications. In this dissertation we uncover the catalytic mechanisms of four enzymes involved in the breakdown of saccharide chains: three via hydrolysis of glycosidic bonds, known as glycoside hydrolases (GHs), and one via sugar phosphorylation (glycoside phosphorylases or GPs), respectively. With computer simulations based in all-atoms models, combining QM/MM approaches with enhanced sampling methods, we simulate the chemical transformations that diverse saccharides undergo in the active site of the enzymes, including their conformational itineraries. The study of enzyme catalysis is an interdisciplinary field, where different techniques from structural biology to biochemistry give insights on their structure and function. Computational techniques such as molecular dynamics, give the opportunity to characterize at the atomic and molecular level states that would be otherwise difficult to capture with experimental methods, such as the short-lived transition states or the sometimes elusive native enzyme-substrate complexes. In this Thesis we aim to uncover the catalytic mechanisms of enzymes relevant for medical and biotechnological applications that have some particularity that sets them apart from other well-established GH mechanisms. This Thesis dissertation contains the following chapters: Chapter 1 - Introduction. Contextualization of the general topics covered in this dissertation, description of the scope of this Thesis and listing of the specific objectives. Chapter 2 - Methods. Section outlining the main theoretical basis of the computational techniques applied and a description of the general workflow used in this work. Chapter 3 - The catalytic mechanism of GH20 lacto-N-biosidase. We uncover the catalytic mechanism of the enzyme LnbB, involved in the degradation of human milk oligosaccharides, which 2 uses the substrate for the nucleophilic role of catalysis. Chapter 4 - The catalytic mechanism of GH130 N-glycan mannoside phosphorylase. We elucidate the catalytic mechanism of the enzyme UhgbMP, which incorporates phosphate to mannosyl substrates via a mechanism involving the substrate in a double proton transfer. Chapter 5 - The catalytic mechanism of GH129 3,6-anhydro-D-galactosidase. We characterize the catalytic mechanism of bacterial ZgGH129, which degrades the unique algal 3,6-anhydro-D-galactosidase-based carrageenan oligosaccharides via an unusual conformational itinerary. Chapter 6- Substrate recognition and catalytic mechanism of human GH29 fucosidase. We elucidate the first reaction step of the reaction mechanism of the enzyme FucA1, recognizes and degrades fucosylated glycans, and unveil structural and dynamic details of its recognition of fucosylated motifs.

[cat] Els carbohidrats constitueixen la família de biomolècules més abundant a la Terra. També coneguts com a glicans, sucres o sacàrids, tenen una estructura polihidroxicarbonílica comuna a la qual s’afegeixen nombroses variacions químiques i estructurals que els confereixen diferents propietats fisicoquímiques. Els carbohidrats participen en nombrosos processos biològics realitzant funcions estructurals, d’emmagatzematge energètic i senyalització molecular. Les formes més simples dels carbohidrats, els monosacàrids, s’uneixen entre si mitjançant enllaços glicosídics per formar estructures més complexes, com els oligo- o polisacàrids, o conjugar-se amb altres famílies de biomolècules, com les glicoproteïnes. La flexibilitat inherent dels carbohidrats, des de les diferents conformacions de les formes cícliques dels monosacàrids a la rotació dels enllaços entre ells, permet diverses disposicions dels seus àtoms, un aspecte que resulta important en els processos en què participen. Els enzims actius en carbohidrats (CAZYmes) catalitzen les reaccions de degradació, síntesi i modificació de carbohidrats. L’estudi d’aquest enzims obre portes per comprendre els processos biològics on estan involucrats, resultant en noves aplicacions mèdiques, biotecnològiques i mediambientals. En aquesta Tesi s’estudia el mecanisme de quatre enzims involucrats en la degradació de cadenes de carbohidrats: tres mitjançant reaccions d’hidròlisi d’enllaços glicosídics (glicosidases o GHs) i un a través de la fosforilació de sucres (glicòsid fosforilases o GPs). Mitjançant simulacions computacionals basades en models atomístics, combinant tècniques QM/MM amb mètodes de mostreig accelerat, simulem les transformacions químiques que experimenten els diferents sacàrids catalitzades pels enzims. L’estudi de la catàlisi enzimàtica és un camp interdisciplinari, on diferents tècniques aporten informació sobre l’estructura i la funció dels enzims. Les tècniques de la química computacional, com la dinàmica molecular, proporcionen una visió atomística dels sistemes i permeten caracteritzar espècies altrament difícils de capturar mitjançant tècniques experimentals, com els estats de transició de les reaccions o els complexos enzim-substrat natius. Els enzims que són objecte d’estudi en aquesta tesi doctoral són la lacto-N‗biosidasa de Bifidobacterium.bifidum?.una mannosa fosforilasa d’un bacteri no identificat del tracte intestinal humà, la 3,6-anhidro-D-galactosidasa de Zobellia.galactanivorans.i la fucosidasa humana 1.