Affinity labeling fatty acyl-CoA synthetase with 9-p-azidophenoxy nonanoic acid and the identification of the fatty acid-binding site (original) (raw)
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Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids
PeerJ, 2015
FadD catalyses the first step in E. coli beta-oxidation, the activation of free fatty acids into acyl-CoA thioesters. This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes. Alcohols and alkanes derived from medium chain fatty acids (MCFAs, 6-12 carbons) are potential biofuels; however, FadD has low activity on MCFAs. Herein, we generate mutations in fadD that enhance its acyl-CoA synthetase activity on MCFAs. Homology modeling reveals that these mutations cluster on a face of FadD from which the co-product, AMP, is expected to exit. Using FadD homology models, we design additional FadD mutations that enhance E. coli growth rate on octanoate and provide evidence for a model wherein FadD activity on octanoate can be enhanced by aiding product exit. These studies provide FadD mutants useful for producing MCFA derivatives and a rationale to alter the substrate specificity of adenylating enzymes.
Journal of Bacteriology, 1998
β-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli , much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This find...
Journal of Biological Chemistry, 2004
We recently reported a new metabolic competency for Escherichia coli, the ability to degrade and utilize fatty acids of various chain lengths as sole carbon and energy sources (Campbell, J. W., Morgan-Kiss, R. M., and Cronan J. E. (2003) Mol. Microbiol. 47, 793-805). This -oxidation pathway is distinct from the previously described aerobic fatty acid degradation pathway and requires enzymes encoded by two operons, yfcYX and ydiQRSTD. The yfcYX operon (renamed fadIJ) encodes enzymes required for hydration, oxidation, and thiolytic cleavage of the acyl chain. The ydiQRSTD operon encodes a putative acyl-CoA synthetase, ydiD (renamed fadK), as well as putative electron transport chain components. We report that FadK is as an acyl-CoA synthetase that has a preference for short chain length fatty acid substrates (<10 C atoms).
Journal of Biological Chemistry, 2002
Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP forming; EC 6.2.1.3) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif (213 YTG-GTTGVAKGA 224 and 356 GYGLTE 361) placing it in the superfamily of adenylate-forming enzymes. A series of sitedirected mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45% wild type activity (fadD G216A , fadD T217A , fadD G219A , and fadD K222A) and 2) those with 10% or less wild-type activity (fadD Y213A , fadD T214A , and fadD E361A). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.
Journal of Biological Chemistry, 2000
Acetyl-CoA carboxylase (ACC) catalyzes the first committed step of the fatty acid synthetic pathway. Although ACC has often been proposed to be a major ratecontrolling enzyme of this pathway, no direct tests of this proposal in vivo have been reported. We have tested this proposal in Escherichia coli. The genes encoding the four subunits of E. coli ACC were cloned in a single plasmid under the control of a bacteriophage T7 promoter. Upon induction of gene expression, the four ACC subunits were overproduced in equimolar amounts. Overproduction of the proteins resulted in greatly increased ACC activity with a concomitant increase in the intracellular level of malonyl-CoA. The effects of ACC overexpression on the rate of fatty acid synthesis were examined in the presence of a thioesterase, which provided a metabolic sink for fatty acid overproduction. Under these conditions ACC overproduction resulted in a 6-fold increase in the rate of fatty acid synthesis.
Proceedings of the National Academy of Sciences, 1976
A soluble enzyme activity which catalyzes the synthesis of acyl-acyl carrier protein from acyl carrier proteins, a long chain fatty acid, and ATP has been demonstrated in E. coli. The reaction requires high concentrations of both Ca++ and Mg++ for activity, and cleaves ATP to AMP and PPi. The fatty acyl product has been identified as acyl-acyl carrier protein by its solubility, thioester linkage, molecular weight, charge, and biological activity. Several criteria indicate the enzyme is distinct from acyl-CoA synthetase. The fatty acid specificity of the enzyme suggests a role of acyl-acyl carrier protein synthetase in the incorporation of fatty acids into phospholipid.
2020
Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the first x-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long timescale molecular dynamics (MD) sim...
Chemistry & Biology, 2010
It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis.