Title: Crystallographic fragment binding studies of the<i>Mycobacterium tuberculosis</i>trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate channeling path between them
Abstract:The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α 2 β 2 tetrameric enzyme in which the α-chain harbors the 2 E -enoyl-CoA hydratase (ECH) and 3 S -hydroxyacyl-CoA dehydrogenase (HAD)...The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α 2 β 2 tetrameric enzyme in which the α-chain harbors the 2 E -enoyl-CoA hydratase (ECH) and 3 S -hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium, and long chain 2 E -enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modelling studies have identified binding sites for the acyl-CoA substrates at the three active sites as well as the NAD + binding pocket at the HAD active site. These studies have also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, in a crystallographic fragment binding study with MtTFE crystals 226 fragments were screened, resulting in the structures of 17 MtTFE-fragment complexes. Analysis of the 143 fragment binding events shows that the ECH active site is the ′binding hotspot′ for the tested fragments, with 50 binding events. The mode of binding of the fragments bound at the active sites provides additional insight on how the long chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 24 fragment binding events between the active sites identify potential transient binding sites of reaction intermediates relevant for possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of these binding sites and to identify substrates for which channeling is crucial.Read More