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The genome of Leishmania major encodes a type II fatty acid biosynthesis pathway for which no structural or biochemical information exists. Here, for the first time, we have characterized the central player of the pathway, the acyl carrier protein (LmACP), using nuclear magnetic resonance (NMR). Structurally, the LmACP molecule is similar to other type II ACPs, comprising a four-helix bundle, enclosing a hydrophobic core. Dissimilarities in sequence, however, exist in helix II (recognition helix) of the protein. The enzymatic conversion of apo-LmACP into the holo form using type I (Escherichia coli AcpS) and type II (Sfp type) phosphopantetheinyl transferases (PPTs) is relatively slow. Mutagenesis studies underscore the importance of the residues present at the protein-protein interaction interface of LmACP in modulating the activity of PPTs. Interestingly, the cognate PPT for this ACP, the L. major 4'-phosphopantetheinyl transferase (LmPPT), does not show any enzymatic activity toward it, though it readily converts other type I and type II ACPs into their holo forms. NMR chemical shift perturbation studies suggest a moderately tight complex between LmACP and its cognate PPT, suggesting inhibition. We surmise that the unique surface of LmACP might have evolved to complement its cognate enzyme (LmPPT), possibly for the purpose of regulation.

We report the use of Coumarin 6 and 1,6-diphenyl-1,3,5-hexatriene (DPH) for the identification of protein aggregates for the first time. The two dyes can be used at very low (nanomolar) concentrations and do not interfere with the aggregation process, as is reported for other commonly used fluorescent protein probes. In the presence of protein aggregates, their quantum yields are significantly high. DPH is able to recognize both amorphous and fibrillar aggregates but cannot distinguish between them. Coumarin 6 can distinguish between both types of aggregates. It also exhibits the characteristic sigmoidal curve of amyloid formation, with higher sensitivity for detection of fibrillation than the conventionally used Thioflavin T.