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.Fatty acids are an essential component of the cellular membranes of all living organisms excepting the Archaea. AcetylCoA carboxylase (ACC) 1 catalyzes the first committed step of the fatty acid synthetic pathway, the formation of malonyl-CoA from acetyl-CoA plus bicarbonate, and ACC has often been postulated to be a rate-controlling step in fatty acid biosynthesis (see, e.g., Refs. 1 and 2). Consistent with this hypothesis, the activity of ACC, the rates of fatty acid synthesis, and the levels of malonyl-CoA are known to be well correlated during hormonal treatments of mammalian tissues (1, 2). However, interpretation of these data is greatly complicated by the recent discovery of a second ACC isoform present in mitochondria (3). Data obtained by use of ACC inhibitors in isolated chloroplasts are also consistent with a regulatory role for ACC in this fatty acid synthetic system (4), although no data on chloroplast malonyl-CoA concentrations were reported. The role of ACC in determining the rate of fatty acid synthesis in vivo seems to remain an open question. As first pointed out by Walsh and Koshland (5), a direct means to approach in vivo pathway regulation is to overproduce candidate enzyme(s) and measure the effect on the flux through the pathway. However, we know of no example in any organism where this approach has been utilized for ACC. A test of the rate-controlling nature of ACC in vivo requires significantly increased levels of ACC activity as well as a metabolic sink (6) for the overproduced fatty acid molecules. Provision of an appropriate sink precludes the possibility that complex lipid synthesis (or the capacity of cell membrane bilayers) could limit the rate of fatty acid synthesis. We have chosen the bacterium Escherichia coli to test if increased ACC activity results in increased rates of fatty acid synthesis. This organism has several experimental advantages. First, the E. coli ACC genes and proteins are well studied (7-13) and the enzyme does not appear to be regulated by small molecules (7). Second, in the presence of high levels of cytosol...
The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.
A 4.8-kb plasmid region carrying the four genes mcjABCDnecessary for production of and immunity to the cyclic peptide antibiotic microcin J25 (MccJ25) has been sequenced. mcjAencodes the primary structure of MccJ25 as a precursor endowed with an N-terminal extension of 37 amino acids. The products ofmcjB and mcjC are thought to be involved in microcin maturation, which implies cleavage of McjA and head-tail linkage of the 21-residue pro-MccJ25. The predicted McjD polypeptide, which is highly similar to several ABC exporters, was found to be required for MccJ25 secretion, thus explaining its ability to confer immunity to MccJ25.
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