A covalent adduct of MbtN, an acyl-ACP dehydrogenase fromMycobacterium tuberculosis, reveals an unusual acyl-binding pocket

Mycobacterium tuberculosis(Mtb) is the causative agent of tuberculosis. Access to iron in host macrophages depends on iron-chelating siderophores called mycobactins and is strongly correlated withMtbvirulence. Here, the crystal structure of anMtbenzyme involved in mycobactin biosynthesis, MbtN, in complex with its FAD cofactor is presented at 2.30 Å resolution. The polypeptide fold of MbtN conforms to that of the acyl-CoA dehydrogenase (ACAD) family, consistent with its predicted role of introducing a double bond into the acyl chain of mycobactin. Structural comparisons and the presence of an acyl carrier protein, MbtL, in the same gene locus suggest that MbtN acts on an acyl-(acyl carrier protein) rather than an acyl-CoA. A notable feature of the crystal structure is the tubular density projecting from N(5) of FAD. This was interpreted as a covalently bound polyethylene glycol (PEG) fragment and resides in a hydrophobic pocket where the substrate acyl group is likely to bind. The pocket could accommodate an acyl chain of 14–21 C atoms, consistent with the expected length of the mycobactin acyl chain. Supporting this, steady-state kinetics show that MbtN has ACAD activity, preferring acyl chains of at least 16 C atoms. The acyl-binding pocket adopts a different orientation (relative to the FAD) to other structurally characterized ACADs. This difference may be correlated with the apparent ability of MbtN to catalyse the formation of an unusualcisdouble bond in the mycobactin acyl chain.

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The genome of a Bacteroidetes inhabitant of the human gut encodes a structurally distinct enoyl-acyl carrier protein reductase (FabI)

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013336 ◽

2020 ◽

Vol 295 (22) ◽

pp. 7635-7652

Author(s):

Christopher D. Radka ◽

Matthew W. Frank ◽

Jiangwei Yao ◽

Jayaraman Seetharaman ◽

Darcie J. Miller ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Change ◽

Conformational Changes ◽

Active Site ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Binding Pocket ◽

Structural Features ◽

Antibacterial Drug ◽

Carrier Protein

Enoyl-acyl carrier protein reductase (FabI) catalyzes a rate-controlling step in bacterial fatty-acid synthesis and is a target for antibacterial drug development. A phylogenetic analysis shows that FabIs fall into four divergent clades. Members of clades 1–3 have been structurally and biochemically characterized, but the fourth clade, found in members of phylum Bacteroidetes, is uncharacterized. Here, we identified the unique structure and conformational changes that distinguish clade 4 FabIs. Alistipes finegoldii is a prototypical Bacteroidetes inhabitant of the gut microbiome. We found that A. finegoldii FabI (AfFabI) displays cooperative kinetics and uses NADH as a cofactor, and its crystal structure at 1.72 Å resolution showed that it adopts a Rossmann fold as do other characterized FabIs. It also disclosed a carboxyl-terminal extension that forms a helix–helix interaction that links the protomers as a unique feature of AfFabI. An AfFabI·NADH crystal structure at 1.86 Å resolution revealed that this feature undergoes a large conformational change to participate in covering the NADH-binding pocket and establishing the water channels that connect the active site to the central water well. Progressive deletion of these interactions led to catalytically compromised proteins that fail to bind NADH. This unique conformational change imparted a distinct shape to the AfFabI active site that renders it refractory to a FabI drug that targets clade 1 and 3 pathogens. We conclude that the clade 4 FabI, found in the Bacteroidetes inhabitants of the gut, have several structural features and conformational transitions that distinguish them from other bacterial FabIs.

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4′-Phosphopantetheine and long acyl chain-dependent interactions are integral to human mitochondrial acyl carrier protein function

MedChemComm ◽

10.1039/c8md00489g ◽

2019 ◽

Vol 10 (2) ◽

pp. 209-220 ◽

Cited By ~ 10

Author(s):

Jaimeen D. Majmudar ◽

Xidong Feng ◽

Nicholas G. Fox ◽

Joseph F. Nabhan ◽

Theresa Towle ◽

...

Keyword(s):

Protein Function ◽

Acyl Carrier Protein ◽

Acyl Chain ◽

Carrier Protein ◽

Acyl Chains

Insights into the role of 4′-PP- and long acyl chains-dependent interactions in human ACPM function.

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Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520042113 ◽

2016 ◽

Vol 113 (7) ◽

pp. 1802-1807 ◽

Cited By ~ 38

Author(s):

Akimasa Miyanaga ◽

Shohei Iwasawa ◽

Yuji Shinohara ◽

Fumitaka Kudo ◽

Tadashi Eguchi

Keyword(s):

Crystal Structure ◽

Protein Interactions ◽

Acyl Carrier Protein ◽

Complex Structure ◽

Binding Pocket ◽

Carrier Protein ◽

Protein Protein Interactions ◽

Substrate Binding Pocket ◽

Insight Into

Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein–protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK–ACP complexes. Because transient enzyme–ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK–ACP complexes, allowing the determination of the crystal structure of the VinK–VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK–VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT.

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[19F]-1H heteronuclear nuclear Overhauser effect studies of the acyl chain-binding site of acyl carrier protein.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)48030-1 ◽

1987 ◽

Vol 262 (19) ◽

pp. 8963-8965

Author(s):

P J Jones ◽

E A Cioffi ◽

J H Prestegard

Keyword(s):

Binding Site ◽

Acyl Carrier Protein ◽

Nuclear Overhauser Effect ◽

Acyl Chain ◽

Carrier Protein ◽

Overhauser Effect ◽

Nuclear Overhauser

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Crystal structure of the Helicobacter pylori enoyl-acyl carrier protein reductase in complex with hydroxydiphenyl ether compounds, triclosan and diclosan

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.21586 ◽

2007 ◽

Vol 69 (3) ◽

pp. 691-694 ◽

Cited By ~ 16

Author(s):

Hyung Ho Lee ◽

Jinho Moon ◽

Se Won Suh

Keyword(s):

Crystal Structure ◽

Helicobacter Pylori ◽

Acyl Carrier Protein ◽

Carrier Protein

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The 1.3-Angstrom-Resolution Crystal Structure of β-Ketoacyl-Acyl Carrier Protein Synthase II from Streptococcus pneumoniae

Journal of Bacteriology ◽

10.1128/jb.185.14.4136-4143.2003 ◽

2003 ◽

Vol 185 (14) ◽

pp. 4136-4143 ◽

Cited By ~ 36

Author(s):

Allen C. Price ◽

Charles O. Rock ◽

Stephen W. White

Keyword(s):

Crystal Structure ◽

Streptococcus Pneumoniae ◽

Active Site ◽

Acyl Carrier Protein ◽

Substrate Binding ◽

Acid Synthesis ◽

Imidazole Ring ◽

Antibacterial Drug ◽

Carrier Protein ◽

Essential Components

ABSTRACT The β-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis. As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery. We have determined the 1.3 Å resolution crystal structure of the β-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organism Streptococcus pneumoniae. The protein adopts a duplicated βαβαβαββ fold, which is characteristic of the thiolase superfamily. The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein. These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily. Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel. The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164. Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg2+ ion which interacts with Glu 346. The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine. This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes.

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