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Structure-Function Relationships in a Glycosyltransferase, a Phosphatase and an Oxidoreductase

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Structure-Function Relationships in a Glycosyltransferase, a Phosphatase and an Oxidoreductase Structure-function relationships in a glycosyltransferase, a phosphatase and an oxidoreductase Susana Margarida Pires Gonçalves Dissertation presented to obtain the Ph.D degree in Biochemistry Instituto de Tecnologia Química e Biológica | Universidade Nova de Lisboa Oeiras, May, 2012 Structure-function relationships in a glycosyltransferase, a phosphatase and an oxidoreductase Susana Margarida Pires Gonçalves Dissertation presented to obtain the Ph.D degree in Biochemistry Instituto de Tecnologia Química e Biológica | Universidade Nova de Lisboa Oeiras, May, 2012 A thesis submitted in conformity with the requirements for the degree of Doctor of Instituto de Tecnologia Química Biológica, Universidade Nova de Lisboa. Supervisor: Dr. Pedro Marques Matias i ii A António e Arcília, meus pais. iii iv ACKOWLEDGMENT I wish to express my sincere gratitude to my supervisor Dr. Pedro Matias for his careful guidance, his extended patience, his invariable availability and most of all his encouragement throughout my doctoral studies. I extend my appreciation to my advisory committee; Dr. Helena Santos and Dr. Peter Donner, and to Dr. Maria Arménia Carrondo, the three of whom provided me with sound advice. Special thanks to Dr. Antony Dean for its valuable recommendations on the elucidation of the catalytic mechanism of -oxidative dehydrogenation by isocitrate dehydrogenase, as well as for his support and encouragement. Dr. Nuno Borges and Dr. Helena Santos, in helpful discussions of the biochemistry behind the “two-step” pathway of mannosylglycerate. I wish to extend my appreciation to my labmates for their friendship and for their precious hints on science and on life. I am profoundly grateful to my parents, my brother and sisters, who played fundamental role in every aspects of my life, and particularly, in my persuit for accomplishing my PhD thesis. v vi THESIS ABSTRACT Enzyme evolution is often constrained by aspects of catalysis. Mechanistically diverse enzymes evolved from a common ancestor still preserve those structural signatures essential to the core chemistry retained by all members of the superfamily. Indeed, these shared features allow superfamilies to be accurately classified, while derived features allow nested families and subfamilies to be identified in a hierarchical fashion. Accurate classification has helped elucidate mechanisms promoting functional diversification, for example catalytic promiscuity, and protein engineering by rational design. Nowadays, a holistic view of enzymes` regulatory mechanisms and catalytic proficiency is provided by the identification of conserved features of molecular architecture in combination with aspects of reaction dynamics. My work focused on the structural elucidation and analysis of three enzymes: a glycosyltransferase; a phosphatase and an oxidorreductase. “Snapshots” along the reaction coordinate of each enzyme were obtained by combining X-ray diffraction with “cryo-trapping” ligand-binding methods. These were used to characterize the molecular mechanisms involved in substrate recognition and binding. They were also used to distinguish between models proposed for the catalytic mechanisms of each enzyme, and provide insights into enzyme dynamics essential for catalysis and the stereo and regio-selective strategies at work. The structural elucidation of the “two-step pathway” of mannosylglycerate (MG) in Thermus thermophilus HB27 was carried out as part of a broader study on the molecular mechanisms of adaptation to extreme environments. MG is a canonical sugar-derivative compatible solute that protects cells against osmotic and thermal stresses. In the first step, mannosyl- 3-phosphoglycerate synthase (MpgS; E.C. 2.4.1.217) catalyses the transfer of the mannosyl-moiety from the activated precursor GDP--D-mannose to the activated nucleophile acceptor D-glycerate-3-phosphate, yielding mannosyl-3- vii phosphoglycerate. In the second step, mannosyl-3-phosphoglycerate is dephosphorylated to MG by mannosyl-3-phosphoglycerate phosphatase (MpgP; E.C. 3.1.3.70). The third enzyme studied is the metal dependent NADP+-linked isocitrate dehydrogenase (IDH; E.C.1.1.1.42) from Escherichia coli. IDH catalyses the oxidative decarboxylation of isocitrate to - ketoglutarate. E. coli IDH is one of the pioneering case-studies of concerted protein dynamics with a productive Michaelis complex. However, a complete picture of these events has been hampered by limitations at the level of crystallographic packing. Main conclusions from each study are: a) Structural elucidation shows T. thermophilus HB27 MpgS is a GT55 glycosyltransferase, a family that falls within the retaining GT-A enzymes (www.cazy.org). Two flexible loops involved in key interactions for the productive binding of substrates were identified by comparing the apo-form with the metal-substrate complex [Mg2+:GDP--D-mannose]. A second metal binding site was found about 6 Å away from the mannose moiety. Kinetic and mutagenesis studies provided evidence that this second metal site is indispensable for catalysis. Additionally, Asp167 of the conserved D-X-D motif was proposed as the catalytic nucleophile in light of the current mechanistic models for the retaining GTs. A survey of enzymes with the GT-A fold and with a bound sugar-donor, or a bound analogue, was used to identify the orientation of the scissile glycosidic bond of the sugar donor with respect to the D/E-X-D motif. The glycosidic bond is oriented towards the motif in the retaining GTs and away from it in the inverting GTs. This feature, which explains the stereochemistry of the reactions, provides a structural signature that will assist efforts in classifying the inverting and retaining GTs. b) Structural elucidation shows T. thermophilus HB27 MpgP is a metal- dependent Haloalcanoic Acid Dehalogenase-like phosphatase (HAD-like) belonging to the Mannosyl-3-Phosphoglycerate Phosphatase family of the Cof- type phosphatases. This structure provides one of the best examples of the combinatorial nature of functional core units, allowing specialization towards viii substrates while preserving core reaction chemistry. The domains of the “open” apo- and the “closed” holo- forms of MpgP are related to each other by a hinge rotation. The dynamics of the catalytic machinery were followed using cryo- trapped reaction species to provide “crystallographic snapshots” along the reaction cycle. Results suggest that phosphoryl-transfer by MpgP from T. thermophilus HB27 involves a concerted DNAN mechanism with Asp8 acting as a catalytic acid in the formation of a short-lived metaphosphate intermediate that is immediately subjected to nucleophilic attack by water. These structures identify the principle mechanistic features of phosphoryl monoester transfer catalysis in members of the HAD superfamily. More generally, they suggest a possible continuum of phosphoryl transfer mechanisms, ranging from those that are purely associative to those that are purely dissociative. c) Comparison of the crystallographic structures of a pseudo-Michaelis- Menten complex of the wildtype EcoIDH, the K100M mutant trapped with its reaction products and several other EcoIDH structures identifies three distinct conformational states: open, quasi-closed and closed. Structural comparisons suggest substrate binding initiates domain closure, with hinge dynamics that span the central -sheet of each monomer in the biological homodimer. Conserved catalytic residues binding the nicotinamide ring of the NADP+ coenzyme and the metal-bound substrate move as rigid bodies together with the hinge rotation. The closed conformations of both the wildtype pseudo- Michaelis ternary complex and the K100M reaction product complex reveal, for the first time, a realistic picture of the mechanistic details of the oxidative - decarboxylation reaction. The structures are entirely in accordance with the postulated roles for the Try160-Lys230* diad in Brønsted acid-base catalysis, as well as revealing the proton relay essential to catalysis. ix x SUMÁRIO DA TESE A evolução enzimática é na maioria dos casos determinada por aspectos associados à catálise. Enzimas que são funcionalmente distintas, mas que partilham uma origem ancestral comum, preservam assinaturas estruturais que asseguram a viabilidade da química base, comum a todos os membros de uma super-família. Estas assinaturas são fundamentais para a classificação de enzimas em diferentes super-famílias, assim como variantes destas assinaturas são indicações para a hierarquização de membros homólogos em famílias e sub-famílias. Tal sistema permite por sua vez a identificação dos mecanismos responsáveis pela diversidade funcional, entre os quais os factores estruturais que favorecem a promiscuidade catalítica e que são relevantes para a indústria biotecnológica e na engenharia de proteínas. Hoje em dia, os conceitos de estrutura e dinâmica estão correlacionados com o de catálise enzimática através da identificação de um conjunto de “movimentos-chave” com uma arquitectura molecular típica. O meu trabalho centrou-se na elucidação da estrutura tridimensional e na análise estrutural de três enzimas: uma glicosiltransferase, uma fosfatase e uma oxidorreductase. Combinando técnicas de difracção de raios-X com métodos de crio-conservação de ligandos com diferentes tempos de incubação nos cristais das enzimas estudadas, obtiveram-se “instantâneos fotográficos” das diferentes etapas das coordenadas de reacção para cada enzima em estudo. A análise destas permitiu a identificação de mecanismos moleculares intervenientes
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