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Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids.

Ford TJ, Way JC - PeerJ (2015)

Bottom Line: This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School , Boston, MA , USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

FadD mutants have increased activity on MCFAs but unaltered affinity for MCFAs.(A) His6-tagged wild-type FadD and the indicated mutants were partially purified via Ni-NTA purification (Materials and Methods) and steady state activity on the indicated fatty acid measured spectrophotometrically using the AMP production assay (Materials and Methods) (Kameda & Nunn, 1981). Vmax (A) and Km (B) values for the indicated substrates. n = 3–4 independent purifications, error bars indicate standard deviation, * indicates p < 0.1 compared to wild-type by two sided students T-test. (C) and (D) Steady state rate of acyl-CoA production using 1.6 µg of Ni-NTA purified, C-terminally His6-tagged wild-type FadD and the indicated mutants with decanoate (C) and oleate (D) as substrates at concentrations roughly 10 times the literature reported Km values in the acyl-CoA production assay (Materials and Methods) (Kameda & Nunn, 1981). n = 3 independent measurements from one or two purifications, error bars indicate standard deviation, and ** indicates p < 0.05 by two sided student’s T-test compared to wild-type FadD.
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fig-3: FadD mutants have increased activity on MCFAs but unaltered affinity for MCFAs.(A) His6-tagged wild-type FadD and the indicated mutants were partially purified via Ni-NTA purification (Materials and Methods) and steady state activity on the indicated fatty acid measured spectrophotometrically using the AMP production assay (Materials and Methods) (Kameda & Nunn, 1981). Vmax (A) and Km (B) values for the indicated substrates. n = 3–4 independent purifications, error bars indicate standard deviation, * indicates p < 0.1 compared to wild-type by two sided students T-test. (C) and (D) Steady state rate of acyl-CoA production using 1.6 µg of Ni-NTA purified, C-terminally His6-tagged wild-type FadD and the indicated mutants with decanoate (C) and oleate (D) as substrates at concentrations roughly 10 times the literature reported Km values in the acyl-CoA production assay (Materials and Methods) (Kameda & Nunn, 1981). n = 3 independent measurements from one or two purifications, error bars indicate standard deviation, and ** indicates p < 0.05 by two sided student’s T-test compared to wild-type FadD.

Mentions: In-vitro assays measuring AMP production by the FadD mutants showed that they have increased activity on MCFAs but not LCFAs. The assay coupled AMP production in the acyl-CoA synthetase reaction to the oxidation of NADH which was monitored spectrophotometrically (Materials and Methods) (Kameda & Nunn, 1981). The Vmax values of the mutants were higher than those of wild-type FadD on octanoate, but were generally lower on oleate (with the exception of mutant H376R). There were no significant changes in the Km toward octanoate for each of the mutants, although the mutant H376R showed an increased Km toward oleate while Y9H had a decreased Km toward oleate (Figs. 3A and 3B). These results indicate that, while the FadD mutations increase the rate of the acyl-CoA synthetase reaction, they do not generally enhance FadD affinity for octanoate.


Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids.

Ford TJ, Way JC - PeerJ (2015)

FadD mutants have increased activity on MCFAs but unaltered affinity for MCFAs.(A) His6-tagged wild-type FadD and the indicated mutants were partially purified via Ni-NTA purification (Materials and Methods) and steady state activity on the indicated fatty acid measured spectrophotometrically using the AMP production assay (Materials and Methods) (Kameda & Nunn, 1981). Vmax (A) and Km (B) values for the indicated substrates. n = 3–4 independent purifications, error bars indicate standard deviation, * indicates p < 0.1 compared to wild-type by two sided students T-test. (C) and (D) Steady state rate of acyl-CoA production using 1.6 µg of Ni-NTA purified, C-terminally His6-tagged wild-type FadD and the indicated mutants with decanoate (C) and oleate (D) as substrates at concentrations roughly 10 times the literature reported Km values in the acyl-CoA production assay (Materials and Methods) (Kameda & Nunn, 1981). n = 3 independent measurements from one or two purifications, error bars indicate standard deviation, and ** indicates p < 0.05 by two sided student’s T-test compared to wild-type FadD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493641&req=5

fig-3: FadD mutants have increased activity on MCFAs but unaltered affinity for MCFAs.(A) His6-tagged wild-type FadD and the indicated mutants were partially purified via Ni-NTA purification (Materials and Methods) and steady state activity on the indicated fatty acid measured spectrophotometrically using the AMP production assay (Materials and Methods) (Kameda & Nunn, 1981). Vmax (A) and Km (B) values for the indicated substrates. n = 3–4 independent purifications, error bars indicate standard deviation, * indicates p < 0.1 compared to wild-type by two sided students T-test. (C) and (D) Steady state rate of acyl-CoA production using 1.6 µg of Ni-NTA purified, C-terminally His6-tagged wild-type FadD and the indicated mutants with decanoate (C) and oleate (D) as substrates at concentrations roughly 10 times the literature reported Km values in the acyl-CoA production assay (Materials and Methods) (Kameda & Nunn, 1981). n = 3 independent measurements from one or two purifications, error bars indicate standard deviation, and ** indicates p < 0.05 by two sided student’s T-test compared to wild-type FadD.
Mentions: In-vitro assays measuring AMP production by the FadD mutants showed that they have increased activity on MCFAs but not LCFAs. The assay coupled AMP production in the acyl-CoA synthetase reaction to the oxidation of NADH which was monitored spectrophotometrically (Materials and Methods) (Kameda & Nunn, 1981). The Vmax values of the mutants were higher than those of wild-type FadD on octanoate, but were generally lower on oleate (with the exception of mutant H376R). There were no significant changes in the Km toward octanoate for each of the mutants, although the mutant H376R showed an increased Km toward oleate while Y9H had a decreased Km toward oleate (Figs. 3A and 3B). These results indicate that, while the FadD mutations increase the rate of the acyl-CoA synthetase reaction, they do not generally enhance FadD affinity for octanoate.

Bottom Line: This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School , Boston, MA , USA.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus