3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus)

Sheldon, Philip S.; Kekwick, R. G. O.; Smith, Colin G.; Sidebottom, C.; Slabas, Antoni R.

doi:10.1016/0167-4838(92)90263-d

Cited by 47 publications

(52 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The K A values for AcAcCoA ranged from 0.6 to 1.2 mM, with the apparent affinity decreasing (K A increasing) as the concentrations of NADPH were increased. The affinity of AcAcCoA to vcFabG in the presence of NADPH was comparable to the affinities of pfFabG (22,25), MabA (23,51), and the FabG from Brassica napus (52). When the concentration of AcAcCoA was held constant and the concentration of NADPH was varied, the binding of NADPH to vcFabG exhibited positive cooperativity with a Hill coefficient of Ϸ2 when the kinetic data were fit to the velocity-based Hill equation (equation 2).…”

Section: Nadph Binding In Wild-type Vcfabgmentioning

confidence: 99%

Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

et al. 2016

View full text Add to dashboard Cite

ABSTRACT␤-Ketoacyl-(acyl carrier protein) reductase (FabG) catalyzes the key reductive reaction in the elongation cycle of fatty acid synthesis (FAS), which is a vital metabolic pathway in bacteria and a promising target for new antibiotic development. The activation of the enzyme is usually linked to the formation of a catalytic triad and cofactor binding, and crystal structures of FabG from different organisms have been captured in either the active or inactive conformation. However, the structural elements which enable activation of FabG require further exploration. Here we report the findings of structural, enzymatic, and binding studies of the FabG protein found in the causative agent of cholera, Vibrio cholerae (vcFabG). vcFabG exists predominantly as a dimer in solution and is able to self-associate to form tetramers, which is the state seen in the crystal structure. The formation of the tetramer may be promoted by the presence of the cofactor NADP(H). The transition between the dimeric and tetrameric states of vcFabG is related to changes in the conformations of the ␣4/␣5 helices on the dimer-dimer interface. Two glycine residues adjacent to the dimer interface (G92 and G141) are identified to be the hinge for the conformational changes, while the catalytic tyrosine (Y155) and a glutamine residue that forms hydrogen bonds to both loop ␤4-␣4 and loop ␤5-␣5 (Q152) stabilize the active conformation. The functions of the aforementioned residues were confirmed by binding and enzymatic assays for the corresponding mutants. IMPORTANCEThis paper describes the results of structural, enzymatic, and binding studies of FabG from Vibrio cholerae (vcFabG). In this work, we dissected the structural elements responsible for the activation of vcFabG. The structural information provided here is essential for the development of antibiotics specifically targeting bacterial FabG, especially for the multidrug-resistant strains of V. cholerae.

show abstract

Section: Nadph Binding In Wild-type Vcfabgmentioning

confidence: 99%

Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Thus, determining the source of reductant for fatty acid synthesis in developing oil seeds is important, and in particular the contribution of NADPH made by the oxidative pentose phosphate pathway (OPPP) 1 to fatty acid synthesis is not known. Of the two reducing steps of fatty acid synthesis, in vitro data indicate that the first (3-ketoacyl-ACP reductase; EC 1.1.1.100) requires NADPH (27), whereas the second (enoyl-ACP reductase, EC 1.3.1.9) requires NADH (28). NADH can be provided by the pyruvate dehydrogenase reaction in plastids, whereas it has long been thought that NADPH for reductive syntheses in nonphotosynthetic plastids is produced by the OPPP (29).…”

mentioning

confidence: 99%

A Flux Model of Glycolysis and the Oxidative Pentosephosphate Pathway in Developing Brassica napus Embryos

Schwender

Ohlrogge

Shachar‐Hill

2003

Journal of Biological Chemistry

242

252

View full text Add to dashboard Cite

Computer simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit metabolic flux parameters to the experimental data. The observed distribution of label in cytosolic and plastidic metabolites indicated that key intermediates of glycolysis and OPPP have similar labeling in these two compartments, suggesting rapid exchange of metabolites between these compartments compared with net fluxes into end products. Cycling between hexose phosphate and triose phosphate and reversible transketolase velocity were similar to net glycolytic flux, whereas reversible transaldolase velocity was minimal. Flux parameters were overdetermined by analyzing labeling in different metabolites and by using data from different labeling experiments, which increased the reliability of the findings. Net flux of glucose through the OPPP accounts for close to 10% of the total hexose influx into the embryo. Therefore, the reductant produced by the OPPP accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis.Brassica napus (rapeseed, canola) is one of the world's major oilseed crops and is also a well studied model for oilseed metabolism (1-21). The main storage compounds in seeds of B. napus are oil (triacylglycerols) and storage proteins, which are derived from sugars and amino acids taken up from the surrounding endosperm liquid (11,12,22,23). Because of its high oil content and ease of genetic transformation, B. napus has also been a target for metabolic engineering of oil metabolism. However, some attempts to engineer plant oils have had limited success (for a review, see Ref. 24). In order to make advances in improving oil yield and quality, a more detailed understanding of metabolism during seed development is needed. In particular, a number of fundamental metabolic issues remain unresolved. These include the source(s) of reductant and ATP for fatty acid synthesis; the degree to which cytosolic, plastidial, and mitochondrial metabolic fluxes are integrated; and the chief metabolic and transport route(s) by which carbon flows from maternal sources to seed storage products.

show abstract

“…Our data show that FabGΔ8 cannot efficiently form dimers, probably due to weakened monomer-monomer interactions at the dimer interface that involve the ␣6/␣7 subdomain and Tyr129=, which is further exacerbated by an increase in temperature and leads to a significant reduction in enzyme activity. Although E. coli FabG crystallized as a tetramer, to our knowledge, the only measurements of the solution structure of a ␤-ketoacyl-ACP reductase were done on a plant enzyme (20). In our study, by size exclusion chromatography, we show that the protein exists predominantly as a dimer in solution.…”

Section: Discussionmentioning

confidence: 73%

An Eight-Residue Deletion in Escherichia coli FabG Causes Temperature-Sensitive Growth and Lipid Synthesis Plus Resistance to the Calmodulin Inhibitor Trifluoperazine

Srinivas

Cronan

2017

J Bacteriol

View full text Add to dashboard Cite

FabG performs the NADPH-dependent reduction of ␤-keto acyl-acyl carrier protein substrates in the elongation cycle of fatty acid synthesis. We report the characterization of a temperature-sensitive mutation (fabGΔ8) in Escherichia coli fabG that results from an in-frame 8-amino-acid residue deletion in the ␣6/␣7 subdomain. This region forms part of one of the two dimerization interfaces of this tetrameric enzyme and is reported to undergo significant conformational changes upon cofactor binding, which define the entrance to the active-site cleft. The activity of the mutant enzyme is extremely thermolabile and is deficient in forming homodimers at nonpermissive temperatures with a corresponding decrease in fatty acid synthesis both in vivo and in vitro. Surprisingly, the fabGΔ8 strain reverts to temperature resistance at a rate reminiscent of that of a point mutant with intragenic pseudorevertants located either on the 2-fold axes of symmetry or at the mouth of the active-site cleft. The fabGΔ8 mutation also confers resistance to the calmodulin inhibitor trifluoperazine and renders the enzyme extremely sensitive to Ca 2ϩ in vitro. We also observed a significant alteration in the lipid A fatty acid composition of fabGΔ8 strains but only in an lpxC background, probably due to alterations in the permeability of the outer membrane. These observations provide insights into the structural dynamics of FabG and hint at yet another point of regulation between fatty acid and lipid A biosynthesis.IMPORTANCE Membrane lipid homeostasis and its plasticity in a variety of environments are essential for bacterial survival. Since lipid biosynthesis in bacteria and plants is fundamentally distinct from that in animals, it is an ideal target for the development of antibacterial therapeutics. FabG, the subject of this study, catalyzes the first cofactor-dependent reduction in this pathway and is active only as a tetramer. This study examines the interactions responsible for tetramerization through the biochemical characterization of a novel temperature-sensitive mutation caused by a short deletion in an important helix-turn-helix motif. The mutant strain has altered phospholipid and lipid A compositions and is resistant to trifluoperazine, an inhibitor of mammalian calmodulin. Understanding its structural dynamics and its influence on lipid A synthesis also allows us to explore lipid homeostasis as a mechanism for antibiotic resistance.KEYWORDS fatty acid synthesis, lipid A, temperature sensitive, antibiotic resistance, calcium, deletion mutant, dimer interface B acteria and plants utilize type II fatty acid synthesis (FAS), a dissociated system of individual enzymes, to generate the entire fatty acid repertoire from acetyl coenzyme A (acetyl-CoA). The intermediates are elongated by a series of four enzymes while

show abstract

3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus)

Cited by 47 publications

References 27 publications

Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

A Flux Model of Glycolysis and the Oxidative Pentosephosphate Pathway in Developing Brassica napus Embryos

An Eight-Residue Deletion in Escherichia coli FabG Causes Temperature-Sensitive Growth and Lipid Synthesis Plus Resistance to the Calmodulin Inhibitor Trifluoperazine

Contact Info

Product

Resources

About