scholarly journals Identification of active site residues implies a two-step catalytic mechanism for acyl-ACP thioesterase

2018 ◽  
Vol 475 (23) ◽  
pp. 3861-3873 ◽  
Author(s):  
Fuyuan Jing ◽  
Marna D. Yandeau-Nelson ◽  
Basil J. Nikolau
Keyword(s):  
Fatty Acid ◽  
Sequence Alignment ◽  
Fatty Acid Synthase ◽  
Catalytic Mechanism ◽  
Fatty Acid Biosynthesis ◽  
Tertiary Structure ◽  
Acyl Carrier Protein ◽  
Cocos Nucifera ◽  
Catalytic Residue ◽  
Thioester Bond

In plants and bacteria that use a Type II fatty acid synthase, isozymes of acyl-acyl carrier protein (ACP) thioesterase (TE) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In the present study, the sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from Cuphea viscosissima and CnFatB2 from Cocos nucifera, resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2, suggests that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the Pseudomonas 4-hydroxybenzoyl-CoA TE, both of which fold in the same hotdog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate.

Binding of NADP+ triggers an open-to-closed transition in a mycobacterial FabG β-ketoacyl-ACP reductase

2017 ◽  
Vol 474 (6) ◽  
pp. 907-921 ◽  
Author(s):  
Mickaël Blaise ◽  
Niël Van Wyk ◽  
Françoise Banères-Roquet ◽  
Yann Guérardel ◽  
Laurent Kremer
Keyword(s):  
Fatty Acid ◽  
Rational Design ◽  
Fatty Acid Synthase ◽  
Catalytic Mechanism ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Reductase Activity ◽  
Protein A ◽  
Structural Features ◽  
Mycobacterial Growth

The ketoacyl-acyl carrier protein (ACP) reductase FabG catalyzes the NADPH/NADH dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the fatty acid biosynthesis elongation cycle. FabG proteins are ubiquitous in bacteria and are part of the type II fatty acid synthase system. Mining the Mycobacterium smegmatis genome uncovered several putative FabG-like proteins. Among them, we identified M. smegmatis MSMEG_6753 whose gene was found adjacent to MSMEG_6754, encoding a recently characterized enoyl-CoA dehydratase, and to MSMEG_6755, encoding another potential reductase. Recombinantly expressed and purified MSMEG_6753 exhibits ketoacyl reductase activity in the presence of acetoacetyl-CoA and NADPH. This activity was subsequently confirmed by functional complementation studies in a fabG thermosensitive Escherichia coli mutant. Furthermore, comparison of the apo and the NADP+-bound MSMEG_6753 crystal structures showed that cofactor binding induces a closed conformation of the protein. A ΔMSMEG_6753 deletion mutant could be generated in M. smegmatis, indicating that this gene is dispensable for mycobacterial growth. Overall, these results showcase the diversity of FabG-like proteins in mycobacteria and new structural features regarding the catalytic mechanism of this important family of enzymes that may be of importance for the rational design of specific FabG inhibitors.

scholarly journals β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis

2000 ◽  
Vol 182 (2) ◽  
pp. 365-370 ◽  
Author(s):  
Keum-Hwa Choi ◽  
Richard J. Heath ◽  
Charles O. Rock
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Chain Fatty Acid ◽  
Carrier Protein ◽  
Acid Biosynthesis ◽  
Biochemical Basis ◽  
E Coli ◽  
Branched Chain

ABSTRACT A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the β-ketoacyl-acyl carrier protein synthase III of Escherichia coli (eFabH) were identified in theBacillus subtilis genome. Their protein products were expressed, purified, and biochemically characterized. Both B. subtilis FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of fatty acid synthesis in vitro with purified E. coli proteins showed that eFabH was the only E. colienzyme incapable of using branched-chain substrates. Expression of either bFabH1 or bFabH2 in E. coli resulted in the appearance of a branched-chain 17-carbon fatty acid. Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production.

scholarly journals Novel inhibitors of the condensing enzymes of the Type II fatty acid synthase of pea (Pisum sativum)

2000 ◽  
Vol 347 (1) ◽  
pp. 205-209 ◽  
Author(s):  
A. Lesley JONES ◽  
Derek HERBERT ◽  
Andrew J. RUTTER ◽  
Jane E. DANCER ◽  
John L. HARWOOD
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Higher Plants ◽  
Acyl Carrier Protein ◽  
Type Ii ◽  
General Activity ◽  
The Individual ◽  
Derivatives Of

The type II fatty acid synthases (FASs) of higher plants (and Escherichia coli) contain three condensing enzymes called β-ketoacyl-ACP synthases (KAS), where ACP is acyl-carrier-protein. We have used novel derivatives of the antibiotic thiolactomycin to inhibit these enzymes. Overall de novo fatty acid biosynthesis was measured using [1-14C]acetate substrate and chloroplast preparations from pea leaves, and [1-14C]laurate was used to distinguish between the effects of the inhibitors on KAS I from those on KAS II. In addition, the activities of these enzymes, together with the short-chain condensing enzyme, KAS III, were measured directly. Six analogues were tested and two, both with extended hydrocarbon side chains, were found to be more effective inhibitors than thiolactomycin. Incubations with chloroplasts and direct assay of the individual condensing enzymes showed that all three compounds inhibited the pea FAS condensing enzymes in the order KAS II > KAS I > KAS III. These results demonstrate the general activity of thiolactomycin and its derivatives against these FAS condensation reactions, and suggest that such compounds will be useful for further detailed studies of inhibition and for use as pharmaceuticals against Type II FASs of pathogens.

scholarly journals 1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

2004 ◽  
Vol 48 (8) ◽  
pp. 3093-3102 ◽  
Author(s):  
Xin He ◽  
Anne McElwee Reeve ◽  
Umesh R. Desai ◽  
Glen E. Kellogg ◽  
Kevin A. Reynolds
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Enzyme Inhibitor ◽  
Acyl Carrier Protein ◽  
Phenyl Group ◽  
Initial Step ◽  
Structural Features ◽  
Inhibitor Complex

ABSTRACT The enzyme FabH catalyzes the initial step of fatty acid biosynthesis via a type II dissociated fatty acid synthase. The pivotal role of this essential enzyme, combined with its unique structural features and ubiquitous occurrence in bacteria, has made it an attractive new target for the development of antibacterial and antiparasitic compounds. We have searched the National Cancer Institute database for compounds bearing structural similarities to thiolactomycin, a natural product which exhibits a weak activity against FabH. This search has yielded several substituted 1,2-dithiole-3-ones that are potent inhibitors of FabH from both Escherichia coli (ecFabH) and Staphylococcus aureus (saFabH). The most potent inhibitor was 4,5-dichloro-1,2-dithiole-3-one, which had 50% inhibitory concentration (IC50) values of 2 μM (ecFabH) and 0.16 μM (saFabH). The corresponding 3-thione analog exhibited comparable activities. Analogs in which the 4-chloro substituent was replaced with a phenyl group were also potent inhibitors, albeit somewhat less effectively (IC50 values of 5.7 and 0.98 μM for ecFabH and saFabH, respectively). All of the 5-chlorinated inhibitors were most effective when they were preincubated with FabH in the absence of substrates. The resulting enzyme-inhibitor complex did not readily regain activity after excess inhibitor was removed, suggesting that a slow dissociation occurs. In stark contrast, a series of inhibitors in which the 5-chloro substituent was replaced with the isosteric and isoelectronic trifluoromethyl group were poorer inhibitors (IC50 values typically ranging from 25 to >100 μM for both ecFabH and saFabH), did not require a preincubation period for maximal activity, and generated an enzyme-inhibitor complex which readily dissociated. Possible modes of binding of 5-chloro-1,2-dithiole-3-ones and 5-chloro-1,2-dithiole-3-thiones with FabH which account for the role of the 5-chloro substituent were considered.

scholarly journals Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis

1997 ◽  
Vol 321 (2) ◽  
pp. 313-318 ◽  
Author(s):  
Elke WINTER ◽  
Monika BRUMMEL ◽  
Ricardo SCHUCH ◽  
Friedrich SPENER
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Condensation Reaction ◽  
Simultaneous Increase ◽  
Carrier Protein ◽  
Acid Biosynthesis ◽  
Cuphea Lanceolata ◽  
Reducing Equivalents

In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalents NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP.

scholarly journals Purification, Characterization, and Identification of Novel Inhibitors of the β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) from Staphylococcus aureus

2002 ◽  
Vol 46 (5) ◽  
pp. 1310-1318 ◽  
Author(s):  
Xin He ◽  
Kevin A. Reynolds
Keyword(s):  
Staphylococcus Aureus ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Genomic Sequence ◽  
Acyl Carrier Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Carrier Protein

ABSTRACT Staphylococcus aureus is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections. The rise of multidrug-resistant strains of S. aureus requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability. We have identified only one fabH in the genome of S. aureus and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of Bacillus subtilis (bFabH1), Escherichia coli (ecFabH), and Mycobacterium tuberculosis (mtFabH). Additional genomic sequence analysis revealed that this S. aureus FabH (saFabH) is not mutated in certain methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains. saFabH was expressed in E. coli with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH. The apparent Km for malonyl-ACP was 1.76 ± 0.40 μM, and the enzyme was active with acetyl-CoA (k cat, 16.18 min−1; Km , 6.18 ± 0.9 μM), butyryl-CoA (k cat, 42.90 min−1; Km , 2.32 ± 0.12 μM), and isobutyryl-CoA (k cat, 98.0 min−1; Km , 0.32 ± 0.04 μM). saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration [IC50], >100 μM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-[1,2]-dithiol-3-one (IC50, 1.87 ± 0.10 μM) and 4-phenyl-5-phenylimino-[1,2,4]dithiazolidin-3-one (IC50, 0.775 ± 0.08 μM).

scholarly journals Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase

2019 ◽  
Author(s):  
Jennifer W. Lou ◽  
Kali R. Iyer ◽  
S. M. Naimul Hasan ◽  
Leah E. Cowen ◽  
Mohammad T. Mazhab-Jafari
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Protein Translocation ◽  
Acyl Carrier Protein ◽  
Reaction Chamber ◽  
Type I ◽  
Acyl Carrier Proteins ◽  
Enoyl Reductase ◽  
Ketoacyl Synthase

ABSTRACTDuring fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

scholarly journals Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jennifer W. Lou ◽  
Kali R. Iyer ◽  
S. M. Naimul Hasan ◽  
Leah E. Cowen ◽  
Mohammad T. Mazhab-Jafari
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Protein Translocation ◽  
Acyl Carrier Protein ◽  
Reaction Chamber ◽  
Type I ◽  
Acyl Carrier Proteins ◽  
Enoyl Reductase ◽  
Ketoacyl Synthase

Abstract During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

scholarly journals Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme

1999 ◽  
Vol 345 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Amine ABBADI ◽  
Monika BRUMMEL ◽  
Burkhardt S. SCHüTT ◽  
Mary B. SLABAUGH ◽  
Ricardo SCHUCH ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Binding Sites ◽  
Fatty Acid Synthase ◽  
Catalytic Mechanism ◽  
Higher Plants ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Type Ii ◽  
Acetyl Coa ◽  
Condensing Enzyme

A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-14C]acetyl-CoA-labelled Cys111, and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys111Ser mutant, however, still was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His261 with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His261. Double mutants Cys111Ser/His261Ala and Cys111Ser/His261Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-14C]malonyl-ACP, a reaction indicating the role of His261 in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg150 and Arg306 in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.

Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA

2018 ◽  
Vol 74 (5) ◽  
pp. 383-393 ◽  
Author(s):  
Tanja Küssau ◽  
Marion Flipo ◽  
Niel Van Wyk ◽  
Albertus Viljoen ◽  
Vincent Olieric ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Crystal Structures ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Rational Drug Design ◽  
Carrier Protein ◽  
Cofactor Binding

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP+-bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP+-bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

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