Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli

Edwards, Patricia C.; Nelsen, Janet; Metz, James G.; Dehesh, Katayoon

doi:10.1016/s0014-5793(96)01437-8

Cited by 87 publications

(105 citation statements)

References 22 publications

(22 reference statements)

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“…Cultures E. coli grown at higher temperatures exhibit significantly lower levels of UFA and an accumulation of palmitoleic acid due to the intrinsic temperature sensitivity of FabF de Mendoza et al 1983;Edwards et al 1997) (Fig. 1) Similar FabF-dependent regulation has been observed in other bacteria (Kutchma et al 1999;Allen and Bartlett 2000).…”

Section: Expression Of L Lactis Fabf Fails To Restore Thermal Contromentioning

confidence: 52%

The Lactococcus lactis FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis

Morgan‐Kiss

Cronan

2008

Arch Microbiol

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The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacylacyl carrier protein synthase. The strain tested was a derivative (called the ΔfabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ΔfabH bypass strain that overproduced FabF and the wild type incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ΔfabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.

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Section: Expression Of L Lactis Fabf Fails To Restore Thermal Contromentioning

confidence: 52%

The Lactococcus lactis FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis

Morgan‐Kiss

Cronan

2008

Arch Microbiol

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“…The KASII-cerulenin complex was prepared as described previously (18). Crystals of the complex were grown by the hanging drop method.…”

Section: Methodsmentioning

confidence: 99%

Structure of the Complex between the Antibiotic Cerulenin and Its Target, β-Ketoacyl-Acyl Carrier Protein Synthase

Moche

Schneider

Edwards³

et al. 1999

Journal of Biological Chemistry

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190

179

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In the biosynthesis of fatty acids, the ␤-ketoacyl-acyl carrier protein (ACP) synthases catalyze chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to either ACP or CoA. The enzyme is a possible drug target for treatment of certain cancers and for tuberculosis. The crystal structure of the complex of the enzyme from Escherichia coli, and the fungal mycotoxin cerulenin reveals that the inhibitor is bound in a hydrophobic pocket formed at the dimer interface. Cerulenin is covalently attached to the active site cysteine through its C2 carbon atom. The fit of the inhibitor to the active site is not optimal, and there is thus room for improvement through structure based design.

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“…As a result, limited substrates would be diverted into both saturated and unsaturated arms of the pathway, resulting in reduced palmitoyl-ACP and palmitoleoyl-ACP concentrations. Increased competition for low levels of palmitoyl-ACP and palmitoleoyl-ACP by phospholipid acyltransferases (PlsB and PlsC) (27,28) would further ensure that only a small proportion of these acyl pools are elongated to stearic and cis-vaccenic acids by FabB and FabF, respectively (29). Although substrate flux into the saturated fatty acid pathway was generally reduced, inhibition of FabA resulted in increased LpxC degradation due to an accumulation of lipid A disaccharide.…”

Section: Excess Substrate Flux Into the Saturated Fatty Acid And Lps mentioning

confidence: 99%

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli

Emiola

Andrews

Heller

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH . Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.lipopolysaccharide | fatty acids | computational model | bacterial membrane regulation

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Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli

Cited by 87 publications

References 22 publications

The Lactococcus lactis FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis

The Lactococcus lactis FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis

Structure of the Complex between the Antibiotic Cerulenin and Its Target, β-Ketoacyl-Acyl Carrier Protein Synthase

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli

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