The Structure of the Holo-Acyl Carrier Protein of Leishmania major Displays a Remarkably Different Phosphopantetheinyl Transferase Binding Interface

Abstract

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.

Mitochondrial Acyl Carrier Protein of Leishmania major Displays Features Distinct from the Canonical Type II ACP

“…enoyl-ACP reductase and 4′-phosphopantetheinyl transferase, were cloned from their genome (Bei Resources). ACPs used in the study were cloned previously . The proteins were induced with 0.4 mM IPTG, and the cells were harvested by centrifugation at 5000 g at 4 °C.…”

“…The purity of the fractions was assessed on a 12% native polyacrylamide gel electrophoresis (PAGE) and 12% sodium dodecyl sulfate–PAGE (SDS–PAGE). The purity of the apo-form was also confirmed by mass spectrometry and C18-reverse phase chromatography as described previously . The pure fractions were pooled and concentrated.…”

“…ACPs used in the study were cloned previously. 39 The proteins were induced with 0.4 mM IPTG, and the cells were harvested by centrifugation at 5000g at 4 °C. Cell pellets were resuspended in 20 mM sodium phosphate buffer, 500 mM NaCl, pH 7.5, followed by sonication and another step of centrifugation at 20,000g at 4 °C.…”

“…The purity of the apo-form was also confirmed by mass spectrometry and C18-reverse phase chromatography as described previously. 39 The pure fractions were pooled and concentrated. The protein was subsequently loaded on a Superdex75 HR 10/300 GL AKTA column, and the single-band pure fractions were pooled.…”

Decrypting the oscillating nature of the 4′‐phosphopantetheine arm in acyl carrier protein AcpM of Mycobacterium tuberculosis

“…In addition to X‐ray crystallography, cryoelectron microscopy has also assisted in the successful determination of the interactions prevalent between ACP and the large megadalton FAS‐I enzyme, but owing to ACP's conformational flexibility, the interaction could not always be correlated with its function . Apart from the above‐mentioned studies, techniques like NMR spectroscopy and molecular dynamics (MD) simulation have been exploited to acquire an in‐depth analysis of the interactions that come into play between ACP and its respective partner enzymes as well as varying length of acyl chains . Generally, in most cases, the short‐lived ACP conformations (< μs) cannot always be resolved based on the Nuclear Overhauser Effect (NOE) pattern of NMR study, making it difficult to obtain a clear picture of ACP dynamics .…”