Backbone and side chain 1H, 15N &amp; 13C chemical shift assignments of the holo-acyl carrier protein (ACP) of Leishmania major

Kumar, Ambrish; Surolia, Avadhesha; Sundd, Monica

doi:10.1007/s12104-012-9362-5

Cited by 3 publications

(4 citation statements)

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“…The 20 lowest-energy structures of the holo-acyl carrier protein of L. major obtained by multidimensional NMR spectroscopy are shown in Figure A. The backbone chemical shift assignments were obtained by acquiring triple-resonance NMR experiments, HNCACB, CBCAcoNH, HNCA, HNcoCA, etc . The side chain chemical shift assignments were derived from CCcoNH-TOCSY and HCCH-TOCSY experiments.…”

Section: Resultsmentioning

confidence: 99%

“…The backbone chemical shift assignments were obtained by acquiring triple-resonance NMR experiments, HNCACB, CBCAcoNH, HNCA, HNcoCA, etc. 32 The side chain chemical shift assignments were derived from CCcoNH-TOCSY and HCCH-TOCSY experiments. The NOESY constraints were obtained from 13 C-edited NOESY, as well as 15 N-edited NOESY experiments.…”

Section: ■ Resultsmentioning

confidence: 99%

Section: Experimental Proceduresmentioning

confidence: 99%

“…Cloning, expression, and purification of ACPs from L. major (LmACP), E. coli, P. falciparum, Mycobacterium tuberculosis, and Homo sapiens were conducted in E. coli as described previously. 32 L. major 4′phosphopantetheinyl transferase (LmPPT, UniProt entry Q4QCW3) was also purified similarly. The proteins were overexpressed by inducing with 0.4 mM isopropyl β-D-1thiogalactopyranoside.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

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

et al. 2015

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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.

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Section: Resultsmentioning

confidence: 99%