Dalwani, S. | Search bioRxiv

bioRxiv2024

Structural enzymological studies of the long chain fatty acyl-CoA synthetase FadD5 from the mce1 operon of Mycobacterium tuberculosis

The cell wall of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is rich in complex lipids. During intracellular stage, Mtb depends on lipids for its survival. Mammalian cell-entry (Mce) 1 complex encoded by the mce1 operon is a mycolic/fatty acid importer. mce1 operon also encodes a putative fatty acyl-CoA synthetase (FadD5; Rv0166), potentially responsible for the activation of fatty acids imported through the Mce1 complex by conjugating them to Coenzyme A. Here we report that FadD5 is associated to membrane although it can be purified as a soluble dimeric protein. ATP and CoA binding influence FadD5s stability and conformation respectively. Enzymatic studies with fatty acids of varying chain lengths show that FadD5 prefers long chain fatty acids as substrates. X-ray crystallographic studies on FadD5 and its variant reveal that the C-terminal domain ([~]100 residues) is cleaved off during crystallization. Noteworthy, deletion of this domain renders FadD5 completely inactive. SAXS studies, however, confirm the presence of full length FadD5 as a dimer in solution. Further structural analysis and comparisons with homologs provide insights on the possible mode of membrane association and fatty acyl tail binding.

biochemistry↗

bioRxiv2024

Crystallographic fragment binding studies of the Mycobacterium tuberculosis 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

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an 2{beta}2 tetrameric enzyme in which the -chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the {beta}-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium, and long chain 2E-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. SynopsisCrystallographic fragment binding studies of the Mycobacterium tuberculosis trifunctional enzyme (MtTFE) have resulted in 143 binding events of 17 fragments out of 226 investigated fragments, suggesting functional sites with respect to substrate binding and substrate channeling.

biochemistry↗