The chain-flipping mechanism of ACP (acyl carrier protein)-dependent enzymes appears universal

2014 ◽  
Vol 460 (2) ◽  
pp. 157-163 ◽  
Author(s):  
John E. Cronan
Keyword(s):  
Fatty Acids ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Protein A ◽  
Serine Residue ◽  
Prosthetic Group ◽  
Acyl Carrier Proteins ◽  
Acyl Chains ◽  
Complex Lipids ◽  
Enzyme Complexes

ACPs (acyl carrier proteins) play essential roles in the synthesis of fatty acids, polyketides and non-ribosomal polypeptides. ACP function requires the modification of the protein by attachment of 4′-phosphopantetheine to a conserved serine residue. The phosphopantetheine thiol acts to tether the starting materials and intermediates as their thioesters. ACPs are small highly soluble proteins composed of four α-helices. The helices form a bundle that acts as a hydrophobic sleeve that sequesters the acyl chains and activated thioesters from solvent. However, in the synthesis of fatty acids and complex lipids the enzymes of the pathway must access the thioester and the proximal carbon atoms in order to perform the needed chemistry. How such access is provided without exposure of the acyl chains to solvent has been a longstanding question due to the lack of acyl-ACP–enzyme complexes, a situation generally attributed to the brevity of the interactions of acyl-ACPs with their cognate enzymes. As discussed in the present review the access question has now been answered by four recent crystal structures, each of which shows that the entire acyl chain plus the 4′-phosphopantetheine prosthetic group partitions from the ACP hydrophobic sleeve into a hydrophobic pocket or groove of the enzyme protein, a process termed chain flipping.

scholarly journals Acyltransferase-mediated selection of the length of the fatty acyl chain and of the acylation site governs activation of bacterial RTX toxins

2020 ◽  
Vol 295 (28) ◽  
pp. 9268-9280 ◽  
Author(s):  
Adriana Osickova ◽  
Humaira Khaliq ◽  
Jiri Masin ◽  
David Jurnecka ◽  
Anna Sukova ◽  
...  
Keyword(s):  
Acyl Chain ◽  
Fatty Acyl ◽  
Biologically Active ◽  
Fatty Acyl Chain ◽  
Acyl Carrier Proteins ◽  
E Coli ◽  
Rtx Toxins ◽  
Wide Range ◽  
Lysine Residues ◽  
Acyl Chains

In a wide range of organisms, from bacteria to humans, numerous proteins have to be posttranslationally acylated to become biologically active. Bacterial repeats in toxin (RTX) cytolysins form a prominent group of proteins that are synthesized as inactive protoxins and undergo posttranslational acylation on ε-amino groups of two internal conserved lysine residues by co-expressed toxin-activating acyltransferases. Here, we investigated how the chemical nature, position, and number of bound acyl chains govern the activities of Bordetella pertussis adenylate cyclase toxin (CyaA), Escherichia coli α-hemolysin (HlyA), and Kingella kingae cytotoxin (RtxA). We found that the three protoxins are acylated in the same E. coli cell background by each of the CyaC, HlyC, and RtxC acyltransferases. We also noted that the acyltransferase selects from the bacterial pool of acyl–acyl carrier proteins (ACPs) an acyl chain of a specific length for covalent linkage to the protoxin. The acyltransferase also selects whether both or only one of two conserved lysine residues of the protoxin will be posttranslationally acylated. Functional assays revealed that RtxA has to be modified by 14-carbon fatty acyl chains to be biologically active, that HlyA remains active also when modified by 16-carbon acyl chains, and that CyaA is activated exclusively by 16-carbon acyl chains. These results suggest that the RTX toxin molecules are structurally adapted to the length of the acyl chains used for modification of their acylated lysine residue in the second, more conserved acylation site.

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

2015 ◽  
Vol 71 (4) ◽  
pp. 862-872 ◽  
Author(s):  
Ai-Fen Chai ◽  
Esther M. M. Bulloch ◽  
Genevieve L. Evans ◽  
J. Shaun Lott ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Double Bond ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Binding Pocket ◽  
Carrier Protein ◽  
Strongly Correlated ◽  
Steady State Kinetics ◽  
Expected Length ◽  
Acyl Chains

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.

scholarly journals Lipid remodelling during epididymal maturation of rat spermatozoa. Enrichment in plasmenylcholines containing long-chain polyenoic fatty acids of the n-9 series

1992 ◽  
Vol 283 (1) ◽  
pp. 235-241 ◽  
Author(s):  
M I Aveldaño ◽  
N P Rotstein ◽  
N T Vermouth
Keyword(s):  
Fatty Acids ◽  
Unsaturated Fatty Acids ◽  
Acyl Chain ◽  
Sperm Maturation ◽  
High Concentration ◽  
Lipid Domain ◽  
Choline Glycerophospholipids ◽  
Epididymal Maturation ◽  
Acyl Chains ◽  
Long Chain

In their transit from the caput to the cauda segments of the epididymis, rat spermatozoa undergo significant modifications in lipid content and composition. The amount of lipid phosphorus per cell decreases, and most lipid classes show specific changes in their constituent fatty acids. A depletion of phosphatidylcholine and phosphatidylethanolamine, concomitant with a virtually unchanged amount of the corresponding plasmalogens, are the major alterations, plasmenylcholine thereby becoming the major phospholipid. Diphosphatidylglycerol, sphingomyelin and the phosphoinositides decrease to a lesser extent or do not change at all, also resulting in relative increases with sperm maturation. Concerning the fatty acids, the proportions of oleate (C18:1, n-9) and linoleate (C18:2, n-6) in most lipids decrease on movement of sperm from caput to cauda, augmenting in turn the proportions of longer-chain (C20 to C24) and more unsaturated fatty acids. Docosapentaenoate (C22:5, n-6) is a major acyl chain present in all lipids at both stages, but uncommon long-chain polyenoic fatty acids of the n-9 series are also present, being almost exclusively found in the choline glycerophospholipids. These fatty acids are found to undergo the most significant changes during sperm maturation. They are minor components of plasmenylcholine in immature spermatozoa, but increase severalfold on maturation, representing more than half of the acyl chains of this major lipid in cells from the cauda. The high concentration of n-9 polyenes in mature sperm plasmenylcholine raises intriguing questions on the possible role epididymal cells may play in providing spermatozoa with such an unusual phospholipid. These plasmenylcholines could contribute to the characteristic lipid domain organization of the mature spermatozoa plasma membrane.

scholarly journals SMc01553 is the sixth acyl carrier protein in Sinorhizobium meliloti 1021

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 230-239 ◽  
Author(s):  
Yadira Dávila-Martínez ◽  
Ana Laura Ramos-Vega ◽  
Sandra Contreras-Martínez ◽  
Sergio Encarnación ◽  
Otto Geiger ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Acyl Carrier Protein ◽  
Duplication Event ◽  
Prosthetic Group ◽  
Acyl Carrier Proteins ◽  
Free Living ◽  
Native Page ◽  
Malonyl Coa ◽  
Recent Duplication

Acyl carrier proteins (ACPs) are required for the transfer of acyl intermediates during fatty acid and polyketide syntheses. In Sinorhizobium meliloti 1021 there are five known ACPs: AcpP, NodF, AcpXL, the ACP domain in RkpA and SMb20651. The genome sequence of S. meliloti 1021 also reveals the ORF SMc01553, annotated as a putative ACP. smc01553 is part of a 6.6 kb DNA region that is duplicated in the chromosome and in the pSymb plasmid, the result of a recent duplication event. SMc01553 overexpressed in Escherichia coli was labelled in vivo with [3H]β-alanine, a biosynthetic building block of the 4′-phosphopantetheine prosthetic group of ACPs. The purified SMc01553 was modified with 4′-phosphopantetheine in the presence of S. meliloti holo-ACP synthase, and this modification resulted in a major conformational change of the protein structure, since the holo-form runs faster in native PAGE than the apo-form. SMc01553 could not be loaded with a malonyl group by malonyl-CoA-ACP transacylase from S. meliloti. Using RT-PCR we could show the presence of mRNA for SMc01553 and of the duplicated ORF SMb22007 in cultures of S. meliloti. However, a mutant in which the two duplicated regions were deleted did not show any different phenotype with respect to the wild-type in the free-living or symbiotic lifestyle.

scholarly journals Bis(monoacylglycerol) phosphate in rat uterine stromal cells: structural characterization and specific esterification of docosahexaenoic acid

2000 ◽  
Vol 351 (3) ◽  
pp. 795-804 ◽  
Author(s):  
Céline LUQUAIN ◽  
René DOLMAZON ◽  
Jean-Marie ENDERLIN ◽  
Christian LAUGIER ◽  
Michel LAGARDE ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Docosahexaenoic Acid ◽  
Polyunsaturated Fatty Acids ◽  
Stromal Cells ◽  
Total Phospholipid ◽  
Acyl Chain ◽  
Primary Alcohol ◽  
High Specificity ◽  
Acyl Chains ◽  
Two Peaks

In rat uterine stromal cells (UIII cells), docosahexaenoic acid (DHA) was esterified extensively in alkenylacyl-glycerophosphoethanolamine and in an unknown phospholipid accounting for only 0.7% of the total phospholipid. The latter was identified as a bis(monoacylglycerol) phosphate (BMP) using MS. Incorporation studies using C18:3n-3 and C20:5n-3 demonstrated that BMP had a high specificity to incorporate DHA and C22 polyunsaturated fatty acids of the (n-3) series. By contrast, polyunsaturated fatty acids of the (n-6) series were never incorporated into BMP. Incubation of UIII cells with 5µM DHA for 24h increased the DHA content of BMP from 36 to 71% of the total acyl chains. [3H]DHA-labelled BMP purified as a single TLC spot was resolved into three peaks using HPLC. These peaks were also observed when cells were labelled with [3H]phosphatidylglycerol, an exogenous BMP precursor, and with [33P]Pi. Electrospray MS of BMP from control cells showed that the first two peaks contained the same molecular species (mainly C22:6n-3/C22:6n-3 and C18:1n-9/C22:6n-3) while the third peak mainly contained the C18:1n-9/C18:1n-9 species. The stereoconfiguration analysis of the compounds revealed an sn-glycero-3-phospho-1′-sn-glycerol configuration for the first peak and sn-glycero-1-phospho-1′-sn-glycerol configurations for the other two. BMP from rat testis was used to establish the positions of the acyl groups. More than 70% of its acyl chains were C22:5n-6. It was separated on HPLC into three peaks that co-migrated with the three peaks of BMP from UIII cells. Lipase activity and NMR analysis of the second peak showed that fatty acids esterified the primary alcohol group on each glycerol moiety. We conclude that the three peaks are stereoisomeric compounds with different acyl-chain locations and may be the result of different metabolic fates depending on subcellular localization.

scholarly journals The elongation of exogenous fatty acids and the control of phospholipid acyl chain length in Micrococcus cryophilus

1980 ◽  
Vol 188 (3) ◽  
pp. 585-592 ◽  
Author(s):  
S P Sandercock ◽  
N J Russell
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Chain Length ◽  
De Novo ◽  
Acyl Chain ◽  
Psychrophilic Bacterium ◽  
Fatty Acid Elongation ◽  
Acyl Chain Length ◽  
Acyl Chains ◽  
Phospholipid Acyl Chain

The synthesis of fatty acids de novo from acetate and the elongation of exogenous satuated fatty acids (C12-C18) by the psychrophilic bacterium Micrococcus cryophilus (A.T.C.C. 15174) grown at 1 or 20 degrees C was investigated. M. cryophilus normally contains only C16 and C18 acyl chains in its phospholipids, and the C18/C16 ratio is altered by changes in growth temperature. The bacterium was shown to regulate strictly its phospholipid acyl chain length and to be capable of directly elongating myristate and palmitate, and possibly laurate, to a mixture of C16 and C18 acyl chains. Retroconversion of stearate into palmitate also occurred. Fatty acid elongation could be distinguished from fatty acid synthesis de novo by the greater sensitivity of fatty acid elongation to inhibition by NaAsO2 under conditions when the supply of ATP and reduced nicotinamide nucleotides was not limiting. It is suggested that phospholipid acyl chain length may be controlled by a membrane-bound elongase enzyme, which interconverts C16 and C18 fatty acids via a C14 intermediate; the activity of the enzyme could be regulated by membrane lipid fluidity.

scholarly journals Molecular basis for the substrate specificity of quorum signal synthases

2017 ◽  
Vol 114 (34) ◽  
pp. 9092-9097 ◽  
Author(s):  
Shi-Hui Dong ◽  
Nicole D. Frane ◽  
Quin H. Christensen ◽  
E. Peter Greenberg ◽  
Rajesh Nagarajan ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Acyl Carrier Protein ◽  
Acyl Donor ◽  
Carrier Protein ◽  
Homoserine Lactones ◽  
Acyl Homoserine Lactones ◽  
Signal Synthesis ◽  
Enzyme Complexes

In severalProteobacteria, LuxI-type enzymes catalyze the biosynthesis of acyl–homoserine lactones (AHL) signals usingS-adenosyl–l-methionine and either cellular acyl carrier protein (ACP)-coupled fatty acids or CoA–aryl/acyl moieties as progenitors. Little is known about the molecular mechanism of signal biosynthesis, the basis for substrate specificity, or the rationale for donor specificity for any LuxI member. Here, we present several cocrystal structures of BjaI, a CoA-dependent LuxI homolog that represent views of enzyme complexes that exist along the reaction coordinate of signal synthesis. Complementary biophysical, structure–function, and kinetic analysis define the features that facilitate the unusual acyl conjugation withS-adenosylmethionine (SAM). We also identify the determinant that establishes specificity for the acyl donor and identify residues that are critical for acyl/aryl specificity. These results highlight how a prevalent scaffold has evolved to catalyze quorum signal synthesis and provide a framework for the design of small-molecule antagonists of quorum signaling.

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

MedChemComm ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 209-220 ◽  
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.

scholarly journals SMb20651 is another acyl carrier protein from Sinorhizobium meliloti

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 257-267 ◽  
Author(s):  
Ana Laura Ramos-Vega ◽  
Yadira Dávila-Martínez ◽  
Christian Sohlenkamp ◽  
Sandra Contreras-Martínez ◽  
Sergio Encarnación ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Sinorhizobium Meliloti ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Fatty Acyl ◽  
Acyl Carrier Proteins ◽  
Coa Ligase ◽  
Acyl Chains

Acyl carrier proteins (ACPs) are small acidic proteins that carry growing acyl chains during fatty acid or polyketide synthesis. In rhizobia, there are four different and well-characterized ACPs: AcpP, NodF, AcpXL and RkpF. The genome sequence of Sinorhizobium meliloti 1021 reveals two additional ORFs that possibly encode additional ACPs. One of these, smb20651, is located on the plasmid pSymB as part of an operon. The genes of the operon encode a putative asparagine synthetase (AsnB), the predicted ACP (SMb20651), a putative long-chain fatty acyl-CoA ligase (SMb20650) and a putative ammonium-dependent NAD+ synthetase (NadE1). When SMb20651 was overexpressed in Escherichia coli, [3H]β-alanine, a biosynthetic building block of 4′-phosphopantetheine, was incorporated into the protein in vivo. The purified SMb20651 was modified with 4′-phosphopantetheine in the presence of S. meliloti holo-ACP synthase (AcpS). Also, holo-SMb20651 was modified in vitro with a malonyl group by malonyl CoA-ACP transacylase. In E. coli, coexpression of SMb20651 together with other proteins such as AcpS and SMb20650 led to the formation of additional forms of SMb20651. In this bacterium, acylation of SMb20651 with C12 : 0 or C18 : 0 fatty acids was detected, demonstrating that this protein is involved in fatty acid biosynthesis or transfer. Expression of SMb20651 was detected in S. meliloti as holo-SMb20651 and acyl-SMb20651.

scholarly journals Divergent acyl carrier protein decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis in malaria parasites

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Seyi Falekun ◽  
Jaime Sepulveda ◽  
Yasaman Jami-Alahmadi ◽  
Hahnbeom Park ◽  
James A Wohlschlegel ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Acid Synthesis ◽  
Complex Iii ◽  
Carrier Protein ◽  
Prosthetic Group ◽  
Malaria Parasites ◽  
Mitochondrial Fatty Acid

Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis and biochemically couple FASII activity to mitochondrial electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast, the mitochondrion of Plasmodium falciparum malaria parasites lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of Nfs1 and the Rieske Fe-S protein in ETC Complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other apicomplexan pathogens, including Toxoplasma and Babesia. This discovery unveils an evolutionary driving force to retain interaction of mitochondrial Fe-S cluster biogenesis with ACP independent of its eponymous function in FASII.

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