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(Cazymes) and Carbohydrate-Binding Modules (Cbms) Involved in Plant Cell Wall Degradation

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(Cazymes) and Carbohydrate-Binding Modules (Cbms) Involved in Plant Cell Wall Degradation UNIVERSIDADE DE LISBOA Faculdade de Medicina Veterinária Structure and Function of Novel Carbohydrate-Active Enzymes (CAZymes) and Carbohydrate-Binding Modules (CBMs) involved in Plant Cell Wall degradation Immacolata Venditto TESE DE DOUTORAMENTO EM CIÊNCIAS VETERINÁRIAS ESPECIALIDADE DE CIÊNCIAS BIOLÓGICAS E BIOMÉDICAS CONSTITUIÇÃO DO JURI ORIENTADOR PRESIDENTE DO JURI: Doutor Carlos Mendes Godinho de Andrade Fontes Reitor da Universidade de Lisboa CO-ORIENTADOR VOGAIS: Doutor Shabir Najmudin Doutor Pedro Maldonado Coutinho Doutor Marco Moracci Doutor Luís Manuel dos Anjos Ferreira Doutor José António Mestre Prates Doutor Carlos Mendes Godinho de Andrade Fontes Doutor Shabir Najmudin 2015 LISBOA ‘There is nothing more wonderful than being a scientist, nowhere I would rather be than in my lab, staining up my clothes and getting paid to play’ (Marie Skłodowska-Curie) Ai miei genitori che sono stati un costante sostegno A mio fratello Angelo per i suoi consigli A Federica Maria per il suo sorriso A Filipe per aver condiviso con me questa esperienza A te Signore che mi sei sempre accanto e mi dai la forza III This work was supported by the European Union Seventh Framework Programme (FP7 2007‐2013) under the WallTraC project (Grant Agreement number 263916). This thesis reflects the author’s views only. The European Community is not liable for any use that may be made of the information contained herein. IV ACKNOWLEDGEMENTS I would like to thank my supervisor Professor Carlos Fontes for giving me the opportunity to do this PhD, for his guidance throughout my years of PhD. I want to thank Dr. Shabir Najmudin for the X-ray crystallography and I am grateful for his patience and support. I would like to thank Professor Luís Ferreira and Professor Victor Alves for their help in my work. I am also grateful for collaborations and I want to thank Professor William Willats for my secondment in Copenhagen and Professor Harry Gilbert for my secondment in Newcastle, for their help and valuable contributions to my work. They were very welcoming and I had a great experience in their lab. I would like to thank Dr. Bernard Henrissat and Professor Pedro Coutinho for their contributions in the project. I would like to thank all members of WallTraC project for their friendship. I want to thank Alexandre Thebaud for the help, support and patience. I would like to thank all the people I have worked with in nutrition lab in FMV: Helena Santos, Joana Brás, Ana Luís, Monica Costa, Kate Cameron, Pedro Bule, Teresa Ribeiro, Vânia Fernandes, Virgínia Pires and Professor Maria Centeno. I would like also to thank D. Maria Paula Silva and Dr. Anabela Gomes for their help and generosity during my years of PhD. I would like to thank Professor Arun Goyal for his help and contributions to my work. V RESUMO Estrutura e Função de novas glucosil hidrolases (CAZymes) e de Módulos de Ligação a Hidratos de Carbono (CMBs) envolvidos na degradação da Parede Celular Vegetal. Os polissacarídeos da parede celular vegetal são uma fonte de energia abundante, eficientemente utilizada por um vasto número de microrganismos, os quais desempenham um papel central na recilagem do carbono. As enzimas secretadas pelos microrganismos aeróbicos, que promovem a hidrólise de hidratos de Carbono (CAZymes), funcionam de froma individualizada, ao passo que as bactérias anaeróbicas organizam essas enzimas num complexo multi-enzimático designado por Celulossoma, o qual efetua uma degradação mais eficiente da parede celular vegetal. As CAZymes são enzimas modulares que contêm, além de domínios catalíticos, módulos de ligação a hidratos de Carbono (CBMs) com função não catalítica. Os CBMs direcionam as enzimas a eles ligadas para os substratos-alvo, potenciando assim a catálise. Neste trabalho mostra-se que os CBMs associado à endoglucanase 5A (EcCel5A) da Eubacterium cellulosolvens designados por CBM65A e CBM65B, possuem uma significativa preferência por xiloglucano. A estrutura tridimensional do CBM65B, em complexo com um derivado oligossacárido do xiloglucano e os estudos de mutagenese realizados no CBM65A, revelaram que o mecanismo de preferência destas proteínas pelo xiloglucano se deve ao estabelecimento de interações hidrofóbicas com as cadeias laterais (xilose) deste substrato (capítulo 2). O genoma da bactéria celulolítica do rúmen Ruminococcus flavifaciens, estirpe FD1, codifica um vasto número de putativas proteínas celulosomais, ainda não estudadas. Neste estudo, os genes que codificam os módulos celulosomais de funções desconhecidas foram clonados e as proteínas por eles codificadas foram expressas em níveis elevados em Escherichia coli. Técnicas complementares, combinando eletroforese em gel nativo, uma plataforma de matriz de alta densidade (microarray) e calorimetria de titulação isotérmica, foram usados para identificar novos CBMs em módulos celulosomais de função desconhecida. Esta estratégia permitiu a identificação de 8 novas famílias de CBMs. Foram determinadas as estruturas tridimensionais representativas de duas destas famílias (CBM-A e CBM-B1), e efectuada a sua caracterização funcional detalhada. O CBM-A e o CBM-B1 apresentam um enrolamento em sanduiche β. O CBM-A liga-se ao β-1,4-glucano ramificado através de uma fenda superficial, revelando preferência por xiloglucano. Em contraste, o CBM-B1 revela uma superfície plana complementar a uma fenda aberta que permite a ligação a uma série de glucanos de tipo β, incluindo o reconhecimento de celulose insolúvel (capítulo 3). Por último, a estrutura do CBM46 derivado de uma endoglucanase do Bacillus halodurans designada por BhCel5B, foi determinada. A BhCel5B é uma enzima multi-modular composta por um domínio catalítico da família GH5_4 no terminal N, seguida por um módulo interno do tipo da imunoglobulina (lg) e o CBM46 no terminal C. O BhCBM46 não se liga a polissacarídeos solúveis ou insolúveis. Porém, a estrutura tridimensional da BhCel5B revelou que o CBM46 é parte integrante da fenda onde se alojam os resíduos responsáveis pela catálise da enzima GH5_4 e, por conseguinte, desempenha um papel importante no reconhecimento do substrato (capítulo 4). Palavras-chave: Enzimas Ativas em Hidratos de Carbono, Módulos de ligação a hidratos de carbono, Glicósido hidrolase, Celulossoma, Ruminococcus flavefaciens VI ABSTRACT Structure and Function of novel Carbohydrate-Active Enzymes (CAZymes) and Carbohydrate Binding Modules (CBMs) involved in Plant Cell Wall degradation. Plant cell wall polysaccharides offer an abundant energy source efficiently utilized by a large repertoire of micro-organisms, which thus play a central role in carbon re-cycling. Aerobic micro-organisms secrete Carbohydrate-Active Enzymes (CAZymes) as free-standing proteins, whereas anaerobic bacteria organize a diverse enzyme consortium in a multi-component complex, the cellulosome, which performs a more efficient deconstruction of this composite structure. CAZymes are modular enzymes containing, in addition to catalytic domains, non-catalytic Carbohydrate-Binding Modules (CBMs). CBMs direct the appended enzymes to their target substrates thus potentiating catalysis. Here we show that the CBMs of Eubacterium cellulosolvens endoglucanase 5A (EcCel5A), designated as CBM65A and CBM65B, display a significant preference for xyloglucan. The crystal structure of CBM65B in complex with a xyloglucan-derived oligosaccharide, in combination with mutagenesis studies on CBM65A, revealed the mechanism by which these proteins display a preference for xyloglucan by establishing hydrophobic interactions with xyloglucan xylose side chains (Chapter 2). The genome of the ruminal cellulolytic bacterium Ruminococcus flavefaciens strain FD-1 encodes a large number of putative novel cellulosomal proteins. Here, genes encoding cellulosomal modules of unknown function were cloned and their corresponding proteins expressed at high levels in Escherichia coli. Complementary techniques combining affinity gel electrophoresis, a microarray platform and isothermal titration calorimetry were used to identify novel CBMs in cellulosomal-modules of unknown function. This strategy allowed the identification of 8 novel CBM families. The structures of representative members of two of these families (CBM-A and CBM-B1) have been solved and detailed functional characterization of these CBMs was performed. CBM-A and CBM-B1 comprise β-sandwich folds. CBM-A binds decorated β-1,4-glucans at a shallow binding cleft and displays preference for xyloglucan. In contrast, CBM-B1 displays a flat surface complementary to an open cleft that allows binding to a range of β-glucans including insoluble cellulose recognition (Chapter 3). Finally, the structure of CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, was solved. BhCel5B is a multi-modular enzyme composed of a GH5_4 N-terminal catalytic domain, followed by an internal immunoglobulin-like module (Ig) and a C-terminal CBM46. BhCBM46 does not bind soluble or insoluble polysaccharides. However, the crystal structure of BhCel5B revealed that CBM46 is integral to the GH5_4 enzyme catalytic cleft and thus plays an important role in substrate recognition (Chapter 4). Key-words: Carbohydrate-Active Enzymes, Carbohydrate-binding module, Glycoside hydrolase, Cellulosome, Ruminococcus flavefaciens. VII INTERNATIONAL PEER-REVIEWED PAPERS This thesis was based on the following manuscripts: Venditto I., Baslé A., Luís A. S., Temple M. J., Ferreira L. M. A., Fontes C. M. G. A., Gilbert H. J. and Najmudin S. (2013) Overproduction, purification, crystallization
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