Molecular dynamics study of hydrolases for saccharification of cellulose and related proteins

Orientador: Munir Salomão SkafTese (doutorado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: A biomassa lignocelulósica proveniente do bagaço de cana-de-açúcar e de outras matérias-primas é um material altamente promissor para a geração de biocombustíveis renováveis e ambientalmente positivos. A melhor opção para a conversão dessa biomassa em açúcares solúveis fermentáveis a etanol, em termos de rendimento e de vantagens ambientais, é a catálise enzimática. Mas esta é também a etapa mais cara do processo de obtenção de etanol de segunda geração devido à baixa eficiência e alto custo dos coquetéis enzimáticos atualmente disponíveis para este fim. Para tornar estes processos mais eficientes e economicamente viáveis, é preciso aprofundar nossa compreensão dos mecanismos de hidrólise celulolítica. Grande investimento em pesquisa tem sido empregado com esta finalidade e, como parte disto, este trabalho consiste em um conjunto de pesquisas desenvolvidas na área de simulação computacional via dinâmica molecular de três enzimas celulolíticas: 1) Laminarinase de Rodhothermus marinus; 2) Endoglucanase 3 de Trichoderma harzianum e 3) b- glicosidase de Aspergillus niger. De modo geral, estes estudos visaram investigar a relação entre o arranjo estrutural e propriedades mensuráveis em laboratório interessantes na avaliação da performance destes biocatalisadores, como afinidade pelo substrato e estabilidade térmica. Como parte do Projeto Temático BioEn, financiado pela FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), estes estudos computacionais foram realizados em estreita colaboração com grupos de biofísicos estruturais e biólogos moleculares, sendo a escolha dos três temas embasada em resultados experimentaisAbstract: The lignocellulosic biomass from sugar cane bagasse and from other raw materials is a highly promising material for the generation of renewable and environmentally positive fuels. In terms of performance and environmental advantages, the best option for converting this biomass into soluble sugars to produce ethanol is the enzymatic catalysis. However, this is also the most expensive step of the second-generation ethanol production due to the low efficiency and high cost of the currently available enzyme cocktails. In order to make the process more efficient and economically viable, it is necessary to deepen the understanding of the cellulolytic hydrolysis mechanisms. Great investment in research has been employed for this purpose, and as part of these efforts, this work consists on a set of molecular dynamics studies of three cellulolytic enzymes, namely: 1) laminarinase from Rodhothermus marinus; 2) Endoglucanase 3 from Trichoderma harzianum and 3) b-glucosidase from Aspergillus niger. In general, these studies aimed to investigate the relationship between the structural arrangement and experimental data that are interesting for the biocatalyst performance evaluation, such as affinity to the substrate and thermal stability. As part of the BioEn Thematic Project, funded by FAPESP (Research Foundation of the State of São Paulo), these computational studies were carried out in close collaboration with structural biophysicists and molecular biologists. The choice of the three proteins considered here was based on these experimental studiesDoutoradoFísico-QuímicaDoutora em Ciência

Molecular dynamics of articaine in POPC membranes

Orientadores: Munir Salomão Skaf, Monica Andrea PickholzDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de QuimicaResumo: Neste trabalho foi feito o estudo das interações da articaína, um anestésico local de ampla aplicação médico-odontológica, com membranas modelo de POPC (palmitoil-oleil-fosfatidilcolina) em condições próximas às biologicamente relevan- tes empregando-se simulações computacionais de dinâmica molecular. Em uma primeira etapa, empregamos métodos quânticos para modelar a articaína com base no campo de força CHARMM. Das simulações de equilíbrio da articaína em POPC, foi possível obter informações como o seu comportamento conformacional e sua posi- ção transversal na bicamada, assim como suas interações especícas com os lipídios. Os estudos foram realizados para os estados neutro e protonado da articaína, consi- derando também seus isômeros ópticos. Estas análises, em conjunto com resultados experimentais de H-RMN realizados pela Prof. Eneida de Paula (IB-UNICAMP) e pelo Prof. Leonardo F. Fraceto (Dpto. de Eng. Ambiental - UNESP, Sorocaba - SP), demonstram que a articaína, em sua forma neutra, posiciona-se preferencial- mente na interface membrana/água, onde interage frequentemente com os lipídios através de ligações de hidrogênio. Através de ferramentas como perfil de densidade eletrônica do sistema, da parte teórica, e perfil do tempo de relaxação longitudinal para diferentes regiões dos lipídios, da parte experimental, foi discutida a lipossolubilidade da articaína em relação a outros anestésicos. Também foram realizadas simulações de não equilíbrio, utilizando a técnica de Dinâmica Molecular de Caminho Induzido, em que uma molécula de articaína foi removida do interior da membrana para o meio aquoso, através de uma força aplicada em seu centro de massa. Com a aplicação da igualdade de Jarzynski a estas simulações, foi possível estimar a energia livre de partição da ATC neutra (forma mais potente) entre os estados em que encontra-se no seio aquoso e no interior da membrana POPC.Abstract: We studied the interactions of articaine - a local anesthetic widely used for me- dical and odontological applications - with model membranes of POPC (palmitoyl-oleyl-phosphatidylcholine) at biological relevant conditions. We have employed molecular dynamics technique, which allowed us to investigate the system at molecular level. Firstly, we applied quantum mechanical methods to parametrize articaine molecule based on CHARMM force field. We have done extensive molceular dynamics simulations, taking into account the different ionization states of the drug (neutral and protonated) as well as its optical isomers. From the equilibirum simulations of articaine in POPC membranes, we investigated the conformational behaviour of the drug, its tranversal position and its specific interactions with the lipids and water molecules. Our results show a preferential orientation of the articaine molecule within the membrane. Neutral articaine was mainly found at the lipid head/water interface, in very good agreement with H-RMN experimental results from Prof. Eneida de Paula (IB-UNICAMP) and Prof. Leonardo F. Fraceto (Dpto. de Eng. Ambiental - UNESP, Sorocaba - SP) and from literature (C. Song et al, 2008). By studying properties like electronic density prole and longitudi- nal time relaxation for different regions of the lipid molecules, we discussed the lipossolubility of articaine in comparison to other local anesthetics. We have also performed non-equilibrium simulations, using steered molecular dynamics (SMD) technique. A single articaine molecule was extracted from the membrane to the wa- ter phase, by applying an external force in its mass centre. Coupling the Jarzynski identity to the SMD simulations, we estimated the partition free energy of the neutral drug (the most potent specie) in POPC membranes.MestradoFísico-QuímicaMestre em Químic

Catalytic mechanism of aryl-ether bond cleavage in lignin by ligf and ligg

Given the abundance of lignin in nature, multiple enzyme systems have been discovered to cleave the β-O-4 bonds, the most prevalent intermonomer linkage. In particular, stereospecific cleavage of lignin oligomers by glutathione S-transferases (GSTs) has been reported in several sphingomonads. Here, we apply quantum mechanics/molecular mechanics simulations to study the mechanism of two glutathione-dependent enzymes in the β-aryl ether catabolic pathway of Sphingomonas sp. SYK-6, namely, LigF, a β-etherase, and LigG, a lyase. For LigF, the free-energy landscape supports a SN2 reaction mechanism, with the monoaromatic leaving group being promptly neutralized upon release. Specific interactions with conserved residues are responsible for stereoselectivity and for activation of the cofactor as a nucleophile. A glutathione conjugate is also released by LigF and serves the substrate of LigG, undergoing a SN2-like reaction, in which Cys15 acts as the nucleophile, to yield the second monoaromatic product. The simulations suggest that the electron-donating substituent at the para-position found in lignin-derived aromatics and the interaction with Tyr217 are essential for reactivity in LigG. Overall, this work deepens the understanding of the stereospecific enzymatic mechanisms in the β-aryl ether cleavage pathway and reveals key structural features underpinning the ligninolytic activity detected in several sphingomonad GSTs123481014210151FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2016/04775-5; 2013/08293-7This work was partially authored by Alliance for Sustainable Energy, LLC, the manager and operator of the National Renewable Energy Laboratory for the U.S. Department of Energy under contract no. DE-AC36-08GO28308. M.F.C. and G.T.B. acknowledge funding for the MD simulations from the U.S. Department of Energy (DOE), Office of Energy Efficiency and Renewable Energy, Bioenergy Technologies Office. G.T.B. also acknowledges funding from the Center for Bioenergy Innovation, a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. E.T.P. and M.S.S. thank funding from the Sao Paulo Research Foundation ̃ (grant nos. 2016/04775-5 and 2013/08293-7). We thank Andreas W. Goetz (San Diego Supercomputer Center, University of California San Diego) who provided DFTB3 implementation in Amber16 for our purposes and for his valuable advices on the adopted methodology. We thank Luiz Carlos Dias (Institute of Chemistry, University of Campinas) for discussions. We acknowledge computer time from the NREL Computational Science Center, which is supported by the DOE Office of Energy Efficiency and Renewable Energy under contract no. DE-AC36-08GO2830

Can exascale computing and explainable artificial intelligence applied to plant biology deliver on the United Nations sustainable development goals?

Human population growth and accelerated climate change necessitate agricultural improvements using designer crop ideotypes (idealized plants that can grow in niche environments). Diverse and highly skilled research groups must integrate efforts to bridge the gaps needed to achieve international goals toward sustainable agriculture. Given the scale of global agricultural needs and the breadth of multiple types of omics data needed to optimize these efforts, explainable artificial intelligence (AI with a decipherable decision making process that provides a meaningful explanation to humans) and exascale computing (computers that can perform 1018 floating-point operations per second, or exaflops) are crucial. Accurate phenotyping and daily-resolution climatype associations are equally important for refining ideotype production to specific environments at various levels of granularity. We review advances toward tackling technological hurdles to solve multiple United Nations Sustainable Development Goals and discuss a vision to overcome gaps between research and policy

Can exascale computing and explainable artificial intelligence applied to plant biology deliver on the United Nations sustainable development goals?

Human population growth and accelerated climate change necessitate agricultural improvements using designer crop ideotypes (idealized plants that can grow in niche environments). Diverse and highly skilled research groups must integrate efforts to bridge the gaps needed to achieve international goals toward sustainable agriculture. Given the scale of global agricultural needs and the breadth of multiple types of omics data needed to optimize these efforts, explainable artificial intelligence (AI with a decipherable decision making process that provides a meaningful explanation to humans) and exascale computing (computers that can perform 1018 floating-point operations per second, or exaflops) are crucial. Accurate phenotyping and daily-resolution climatype associations are equally important for refining ideotype production to specific environments at various levels of granularity. We review advances toward tackling technological hurdles to solve multiple United Nations Sustainable Development Goals and discuss a vision to overcome gaps between research and policy

A mini-TGA protein modulates gene expression through heterogeneous association with transcription factors

TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirmed that StTGA2.1 overexpression compensates for SA-deficiency, indicating a distinct mechanism of action compared with model plant species. To understand the underlying mechanisms, we showed that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigated the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirmed that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases (StPRX), which are known to play role in immune response. Finally, via structural modeling and molecular dynamics simulations, we hypothesized that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to distinct functions and that such events should be studied carefully in other protein families