RESUMO
α/ß hydrolases make up a large and diverse protein superfamily. In natural product biosynthesis, cis-acting thioesterase α/ß hydrolases can terminate biosynthetic assembly lines and release products by hydrolyzing or cyclizing the biosynthetic intermediate. Thioesterases can also act in trans, removing aberrant intermediates and restarting stalled biosynthesis. Knockout of this "editing" function leads to reduced product titers. The borrelidin biosynthetic gene cluster from Streptomyces parvulus Tü4055 contains a hitherto uncharacterized stand-alone thioesterase, borB. In this work, we demonstrate that purified BorB cleaves acyl substrates with a preference for propionate, which supports the hypothesis that it is also an editing thioesterase. The crystal structure of BorB shows a wedgelike hydrophobic substrate binding crevice that limits substrate length. To investigate the structure-function relationship, we made chimeric BorB variants using loop regions from characterized homologues with different specificities. BorB chimeras slightly reduced activity, arguing that the modified region is a not major determinant of substrate preference. The structure-function relationships described here contribute to the process of elimination for understanding thioesterase specificity and, ultimately, engineering and applying trans-acting thioesterases in biosynthetic assembly lines.
Assuntos
Proteínas de Bactérias/química , Ácido Graxo Sintases/química , Streptomyces/enzimologia , Tioléster Hidrolases/química , Proteínas de Bactérias/genética , Domínio Catalítico , Ácido Graxo Sintases/genética , Cinética , Família Multigênica , Engenharia de Proteínas , Especificidade por Substrato , Tioléster Hidrolases/genéticaRESUMO
In the search for molecular machinery for custom biosynthesis of valuable compounds, the modular type I polyketide synthases (PKSs) offer great potential. In this study, we investigate the flexibility of BorM5, the iterative fifth module of the borrelidin synthase, with a panel of non-native priming substrates in vitro. BorM5 differentially extends various aliphatic and substituted substrates. Depending on substrate size and substitution BorM5 can exceed the three iterations it natively performs. To probe the effect of methyl branching on chain length regulation, we engineered a BorM5 variant capable of incorporating methylmalonyl- and malonyl-CoA into its intermediates. Intermediate methylation did not affect overall chain length, indicating that the enzyme does not to count methyl branches to specify the number of iterations. In addition to providing regulatory insight about BorM5, we produced dozens of novel methylated intermediates that might be used for production of various hydrocarbons or pharmaceuticals. These findings enable rational engineering and recombination of BorM5 and inform the study of other iterative modules.