Characterization of the β-Carbon Processing Reactions of the Mammalian Cytosolic Fatty Acid Synthase:  Role of the Central Core

Witkowski, Andrzej; Joshi, and Anil K.; Smith, Stuart

doi:10.1021/bi048988n

Cited by 33 publications

(42 citation statements)

References 31 publications

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“…Indeed, the original assignment of Lys-1699 to the ER enzyme was based on the observation that PLP labeling selectively inhibited the ER activity while leaving the other partial reactions unaffected. In addition, Smith and co-workers (34,35) mutated residues in the proposed NADPHbinding sites of the ER (Gly-1672 and Gly-1673) and BKR (Gly-1886 and Gly-1888) enzymes of rat FAS, and they demonstrated that these mutations had selective effects on the two partial activities. Given the extensive data for two NADPH-binding sites, our current hypothesis is that NADPH binding to one cofactor site is sensed by the second cofactor site and, thus, that the result of mutating Lys-1699 has an allosteric effect on the BKR reaction.…”

Section: Discussionmentioning

confidence: 99%

Substrate Recognition by the Human Fatty-acid Synthase

Carlisle-Moore

Gordon²,

Machutta

et al. 2005

Journal of Biological Chemistry

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The human fatty-acid synthase (HFAS) is a potential target for anti-tumor drug discovery. As a prelude to the design of compounds that target the enoyl reductase (ER) component of HFAS, the recognition of NADPH and exogenous substrates by the ER active site has been investigated. Previous studies demonstrate that modification of Lys-1699 by pyridoxal 5-phosphate results in a specific decrease in ER activity. For the overall HFAS reaction, the K1699A and K1699Q mutations reduced k cat and k cat /K NADPH by 8-and 600-fold, respectively (where K NADPH indicates the K m value for NADPH). Thus, Lys-1699 contributes 4 kcal/mol to stabilization of the rate-limiting transition state following NADPH binding, while also stabilizing the most stable ground state after NADPH binding by 3 kcal/mol. A similar effect of the mutations on the ER partial reaction was observed, in agreement with the proposal that Lys-1699 is located in the ER NADPH-binding site. Most unexpectedly, however, both k cat and k cat /K NADPH for the ␤-ketoacyl reductase (BKR) reaction were also impacted by the Lys-1699 mutations, raising the possibility that the ER and BKR activities share a single active site. However, based on previous data indicating that the two reductase activities utilize distinct cofactor binding sites, mutagenesis of Lys-1699 is hypothesized to modulate BKR activity via allosteric effects between the ER and BKR NADPH sites.Fatty acid synthesis generates important intermediates for the construction of cell membranes and for energy storage (1). In humans the reactions resulting in the production of fatty acids are catalyzed by a multifunctional enzyme complex (HFAS), 3 consisting of the following seven catalytic activities: acetyl/malonyl transacylase, ␤-ketoacyl synthase, ␤-ketoacyl reductase (BKR), ␤-hydroxyacyl dehydratase (DH), enoyl reductase (ER), and thioesterase (2-10). In addition, there is an acyl carrier protein (ACP) domain, which carries fatty acyl intermediates in the form of fatty acyl thioesters (5). Palmitate, a C 16 -saturated fatty acid, is the major product produced (5, 11). Fatty acid synthesis, although important evolutionarily for survival during famines (1), was thought to have very little significance for today's modern society (1,9,10,12). This is because of the dietary intake of lipids, which causes HFAS to be down-regulated in normal cells (6,7,13,14). However, HFAS overexpression in many cancers, such as in breast, prostate, and colon tumors, and also in many pre-malignant growths, has led to the hypothesis that HFAS is an important target for the study of tumor biology (7,13,14).Our laboratory has a long standing interest in the development of antibacterial compounds that target prokaryotic fatty acid synthesis. Thus, an initial interest in HFAS was as a control system for validating the specificity of our bacterial FAS inhibitors. However, as described above, HFAS is now thought to be a bona fide target in its own right as a putative anti-tumor target. There have also been suggestions that HFAS inhibi...

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Section: Discussionmentioning

confidence: 99%

Substrate Recognition by the Human Fatty-acid Synthase

Carlisle-Moore

Gordon²,

Machutta

et al. 2005

Journal of Biological Chemistry

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“…16a,33 Each KR 0 protein, as well as redox-active EryKR1 as positive control and NADPH-nonbinding EryKR3 0 as negative control, was prepared at 10 μM in 50 mM sodium phosphate, pH 7.2. Solutions of NADPH (10-μL) of increasing concentration were added to the microplate wells containing 90 μL of purified 10 μM KR protein solution or buffer alone.…”

Section: Experimental Methodsmentioning

confidence: 99%

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis

et al. 2017

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The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR0) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP+ in the presence of redox-inactive, recombinant NanKR10 or NanKR50, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR70 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR0-catalyzed isotope exchange of the reversibly-generated, transiently-formed oxidation product [2-2H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.

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“…To avoid loss of short fatty acids during evaporation, fatty acids were converted to the potassium salts, solvent was removed by evaporation with nitrogen and the fatty acid salts were derivatized with phenacyl-8 (Pierce). Phenacyl esters were analyzed by reversed-phase HPLC (22). To improve separation of phenacyl esters of short chain fatty acids and lipoic acid, the gradient was modified as follow: 45% solvent B (acetonitrile) for 5 min, then linear gradient to 56% B over 13 min, linear gradient to 90% B over 12 min, and finally linear gradient to 98% B over 9 min.…”

Section: Isolation Purification and Subfractionation Of Bovine Heartmentioning

confidence: 99%

Coupling of the de Novo Fatty Acid Biosynthesis and Lipoylation Pathways in Mammalian Mitochondria

Witkowski¹,

Joshi²,

Smith³

2007

Journal of Biological Chemistry

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The objective of this study was to identify the products and possible role of a putative pathway for de novo fatty acid synthesis in mammalian mitochondria. Bovine heart mitochondrial matrix preparations were prepared free from contamination by proteins from other subcellular components and, using a combination of radioisotopic labeling and mass spectrometry, were shown to contain all of the enzymes required for the extension of a 2-carbon precursor by malonyl moieties to saturated acyl-ACP thioesters containing up to 14 carbon atoms. A major product was octanoyl-ACP and, in the presence of the apo-H-protein of the glycine cleavage complex, the newly synthesized octanoyl moieties were translocated to the lipoylation site on the acceptor protein. These studies demonstrate that one of the functions of the de novo fatty acid biosynthetic pathway in mammalian mitochondria is to provide the octanoyl precursor required for the essential protein lipoylation pathway.Lipoic acid was discovered in the early 1950s, through a collaboration between Lester Reed and Eli Lilly and Company (1), and initially was thought to represent a new member of the B vitamin family. Thirty years elapsed before evidence was obtained indicating that lipoic acid was not a vitamin for animals and that radiolabel from acetate and octanoate could be incorporated into lipoic acid, most likely in the liver (2). However, the mechanism by which lipoic acid is synthesized in animals has yet to be elucidated. In addition to performing an important role as an antioxidant in the free acid form (3), lipoyl moieties are essential cofactors for several mitochondrial multienzyme complexes that play critical roles in energy metabolism, including the ␣-ketoacid dehydrogenases and the glycine cleavage system. In each of these enzyme complexes, lipoyl moieties are covalently attached via an amide bond to the ⑀-amino group of specific lysine residues (4). Studies in prokaryotes have established that lipoyl moieties can be derived either from endogenous lipoic acid, via a lipoyl-AMP intermediate formed by the lipoyl ligase, LplA (5), or from octanoyl-ACP, 2 an intermediate in fatty acid synthesis, by the insertion of sulfur atoms at C-6 and C-8 in a reaction catalyzed by lipoic acid synthase, LipA (6 -8); insertion of the sulfur atoms can occur prior to, or following, transfer from ACP thioester linkage to the lysine residue on the acceptor protein in a reaction catalyzed by lipoyl transferase, LipB. Homologs of LplA, LipA, and LipB have been identified in eukaryotes (9 -11), suggesting that the same two pathways for production of lipoyl moieties may be operative in mitochondria. Recently, strong evidence has been obtained for the existence of a mitochondrial pathway for de novo fatty acid biosynthesis in both fungi (12) and plants (13,14) and several of the enzymes required for the de novo synthesis of fatty acids by a putative type II "ACP track" pathway have been identified and characterized in humans and shown to be nuclear-encoded, mitochondrially targeted pro...

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Characterization of the β-Carbon Processing Reactions of the Mammalian Cytosolic Fatty Acid Synthase: Role of the Central Core

Cited by 33 publications

References 31 publications

Substrate Recognition by the Human Fatty-acid Synthase

Substrate Recognition by the Human Fatty-acid Synthase

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis

Coupling of the de Novo Fatty Acid Biosynthesis and Lipoylation Pathways in Mammalian Mitochondria

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