RESUMO
The study of non-natural biocatalytic transformations relies heavily on empirical methods, such as directed evolution, for identifying improved variants. Although exceptionally effective, this approach provides limited insight into the molecular mechanisms behind the transformations and necessitates multiple protein engineering campaigns for new reactants. To address this limitation, we disclose a strategy to explore the biocatalytic reaction space and garner insight into the molecular mechanisms driving enzymatic transformations. Specifically, we explored the selectivity of an "ene"-reductase, GluER-T36A, to create a data-driven toolset that explores reaction space and rationalizes the observed and predicted selectivities of substrate/mutant combinations. The resultant statistical models related structural features of the enzyme and substrate to selectivity and were used to effectively predict selectivity in reactions with out-of-sample substrates and mutants. Our approach provided a deeper understanding of enantioinduction by GluER-T36A and holds the potential to enhance the virtual screening of enzyme mutants.
Assuntos
Ciência de Dados , Ciência de Dados/métodos , Biocatálise , Estereoisomerismo , Especificidade por Substrato , Ligantes , Mutação , Modelos MolecularesRESUMO
Ribosomally synthesized post-translationally modified peptides (RiPPs) are ubiquitous and represent a structurally diverse class of natural products. The ribosomally encoded precursor polypeptides are often extensively modified post-translationally by enzymes that are encoded by coclustered genes. Radical S-adenosyl-l-methionine (SAM) enzymes catalyze numerous chemically challenging transformations. In RiPP biosynthetic pathways, these transformations include the formation of C-H, C-C, C-S, and C-O linkages. In this paper, we show that the Geobacter lovleyi sbtM gene encodes a radical SAM protein, SbtM, which catalyzes the cyclization of a Cys/SeCys residue in a minimal peptide substrate. Biochemical studies of this transformation support a mechanism involving H-atom abstraction at the C-3 of the substrate Cys to initiate the chemistry. Several possible cyclization products were considered. The collective biochemical, spectroscopic, mass spectral, and computational observations point to a thiooxazole as the product of the SbtM-catalyzed modification. To our knowledge, this is the first example of a radical SAM enzyme that catalyzes a transformation involving a SeCys-containing peptide and represents a new paradigm for formation of oxazole-containing RiPP natural products.
Assuntos
Peptídeos Antimicrobianos/metabolismo , Geobacter/metabolismo , S-Adenosilmetionina/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/fisiologia , Peptídeos Antimicrobianos/fisiologia , Produtos Biológicos/metabolismo , Catálise , Geobacter/patogenicidade , Espectrometria de Massas/métodos , Oxazóis , Processamento de Proteína Pós-Traducional/fisiologia , Proteômica/métodos , Ribossomos , S-Adenosilmetionina/metabolismoRESUMO
Methylammonium lead halide perovskite-based solar cells have demonstrated efficiencies as high as 24.2 %, highlighting their potential as inexpensive and solution-processable alternatives to silicon solar cell technologies. Poor stability towards moisture, ultraviolet irradiation, heat, and a bias voltage of the perovskite layer and its various device interfaces limits the commercial feasibility of this material for outdoor applications. Herein, we investigate the role of hydrogen bonding interactions induced when metal halide perovskite crystals are crosslinked with alkyl or π-conjugated boronic acid small molecules (-B(OH)2 ). The crosslinked perovskite crystals are investigated under continuous light irradiation and moisture exposure. These studies demonstrate that the origin of the interaction between the alkyl or π-conjugated crosslinking molecules is due to hydrogen bonding between the -B(OH)2 terminal group of the crosslinker and the I of the [PbI6 ]4- octahedra of the perovskite layer. Also, this interaction influences the stability of the perovskite layer towards moisture and ultraviolet light irradiation. Morphology and structural analyses, as well as IR studies as a function of aging under both dark and light conditions show that π-conjugated boronic acid molecules are more effective crosslinkers of the perovskite crystals than their alkyl counterparts thus imparting better stability towards light and moisture degradation.