Protein–protein interaction based substrate control in the <i>E. coli</i> octanoic acid transferase, LipB

Bartholow, Thomas G.; Sztain, Terra; Young, Megan; Davis, Thomas B.; Abagyan, Ruben; Burkart, Michael D.

doi:10.1039/d1cb00125f

Cited by 6 publications

(2 citation statements)

References 33 publications

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“…coli ( Ec ACP) acyl-ACP intermediates have been characterized previously using NMR and X-ray crystallography (PDB 2FAE, 2FAC). , Most of the information about the canonical type II acyl-ACP intermediates has been gained from those studies.…”

Section: Resultsmentioning

confidence: 99%

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

Dhembla,

Kumar,

Arya

et al. 2023

Biochemistry

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Prokaryotes synthesize fatty acids using a type II synthesis pathway (FAS). In this process, the central player, i.e., the acyl carrier protein (ACP), sequesters the growing acyl chain in its internal hydrophobic cavity. As the acyl chain length increases, the cavity expands in size, which is reflected in the NMR chemical shift perturbations and crystal structures of the acyl-ACP intermediates. A few eukaryotic organelles, such as plastids and mitochondria, also harbor type II fatty acid synthesis machinery. Plastid FAS from spinach and Plasmodium falciparum has been characterized at the molecular level, but the mitochondrial pathway remains unexplored. Here, we report NMR studies of the mitochondrial acyl–acyl carrier protein intermediates of Leishmania major (acyl-LmACP). Our studies show that LmACP experiences remarkably small conformational changes upon acylation, with perturbations confined to helices II and III only. CastP determined that the cavity size of apo-LmACP (PDB entry 5ZWT) is less than that of Escherichia coli ACP (PDB 1T8K). Thus, the small chemical shift perturbations observed in the LmACP intermediates, coupled with CastP results, suggest an unusually small cavity when fully expanded. The faster rate of C8-LmACP chain hydrolysis compared to E. coli ACP (EcACP) also supports these convictions. Structure comparison of LmACP with other type II ACP disclosed unique differences in the helix I and loop I conformations, as well as several residues present there. Numerous hydrophobic residues in helix I and loop I (conserved in all mitochondrial ACPs) are substituted with hydrophilic residues in the bacterial/plastid type II ACP. For instance, Phe and leucine at positions 14 and 34 in LmACP are substituted with a hydrophilic residue and Ala in bacterial/plastid type II ACP. Mutation of Leu 34 to Ala (corresponding residue in EcACP) resulted in a complete loss of structure, underscoring its importance in maintaining the ACP fold. Thus, our NMR studies, combined with insights from the crystal structure, highlight several unique features of LmACP, distinct from the prokaryote and plastid type II ACP. Given the high sequence identity, the features might be conserved in all mitochondrial ACPs.

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Section: Resultsmentioning

confidence: 99%

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

Dhembla,

Kumar,

Arya

et al. 2023

Biochemistry

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“…91 Recent works on LipB, an enzyme that directs C8-ACP to lipoic acid biosynthesis, elucidates an allostery that is chain length dependent. 101,107 Acyl chain terminal carbon 13 C-labeled C8 and C12-ACP, which chain-flipping event can be tracked by HSQC NMR, are titrated with active site cysteine-mutated LipB, and only the cognate C8-ACP performs chain-flipping. 101 This study provides the first solid evidence of allosteric regulation of EcACP.…”

Section: Allosteric Regulationmentioning

confidence: 99%

Enzymology of standalone elongating ketosynthases

Jiang

Burkart

2022

Chem. Sci.

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Using NMR Titration Experiments to Study E. coli FAS-II- and AcpP-Mediated Protein–Protein Interactions

Mellor,

Sanlley,

Burkart

2023

Methods in Molecular Biology

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Protein–protein interaction based substrate control in the E. coli octanoic acid transferase, LipB

Abstract: Lipoic acid is an essential cofactor produced in all organisms by diverting octanoic acid derived as an intermediate of type II fatty acid biosynthesis. In bacteria, octanoic acid is transferred...

Cited by 6 publications

References 33 publications

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

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

Enzymology of standalone elongating ketosynthases

Using NMR Titration Experiments to Study E. coli FAS-II- and AcpP-Mediated Protein–Protein Interactions

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