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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

Rationally designed, site directed FadD mutants increase E. coli ΔfadR growth rate on octanoate when compared to wild-type FadD.(A) FadD homology model generated using The SWISS-MODEL Homology modeling server (Arnold et al., 2006; Benkert, Biasini & Schwede, 2011; Biasini et al., 2014) and the Thermus thermophilus structure as the template. The model was visualized in PyMOL with large N-terminal domain in gray, smaller C-terminal domain in white, and myristoyl-AMP (overlayed from the Thermus thermophillus structure) in yellow (myristoyl group) and magenta (AMP) (Hisanaga et al., 2004). Residues whose mutation results in increased growth rate on octanoate are color-coded according to the identity of the mutation (text below model, Y9H and V4F W5L are excluded from the model). (B) (i) Surface representation of the FadD homology model with residues mutated to glycine in (ii) shown in blue (mutations that decrease growth rate compared to wild-type) and red (mutations that increase growth rate compared to wild-type). (ii) Percent increase in exponential growth rate compared to wild-type FadD caused by mutating the residues on the X-axis to glycine. (C) (i) Cartoon representation of FadD homology model with residues mutated in (ii) in red. (ii) Percent increase in exponential growth rate compared to wild-type FadD caused by the FadD mutations depicted on the X-axis. n = 13–18, error bars indicate standard error in all cases, ** indicates growth rate significantly different from wild-type with p < 0.05 by two-sided students T-test.
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fig-4: Rationally designed, site directed FadD mutants increase E. coli ΔfadR growth rate on octanoate when compared to wild-type FadD.(A) FadD homology model generated using The SWISS-MODEL Homology modeling server (Arnold et al., 2006; Benkert, Biasini & Schwede, 2011; Biasini et al., 2014) and the Thermus thermophilus structure as the template. The model was visualized in PyMOL with large N-terminal domain in gray, smaller C-terminal domain in white, and myristoyl-AMP (overlayed from the Thermus thermophillus structure) in yellow (myristoyl group) and magenta (AMP) (Hisanaga et al., 2004). Residues whose mutation results in increased growth rate on octanoate are color-coded according to the identity of the mutation (text below model, Y9H and V4F W5L are excluded from the model). (B) (i) Surface representation of the FadD homology model with residues mutated to glycine in (ii) shown in blue (mutations that decrease growth rate compared to wild-type) and red (mutations that increase growth rate compared to wild-type). (ii) Percent increase in exponential growth rate compared to wild-type FadD caused by mutating the residues on the X-axis to glycine. (C) (i) Cartoon representation of FadD homology model with residues mutated in (ii) in red. (ii) Percent increase in exponential growth rate compared to wild-type FadD caused by the FadD mutations depicted on the X-axis. n = 13–18, error bars indicate standard error in all cases, ** indicates growth rate significantly different from wild-type with p < 0.05 by two-sided students T-test.

Mentions: FadD homology models generated using the SWISS-MODEL Homology modeling server (Arnold et al., 2006; Benkert, Biasini & Schwede, 2011; Biasini et al., 2014) and the Thermus thermophilus structure as the template, the I-TASSER server (Roy, Kucukural & Zhang, 2010; Yang et al., 2015; Zhang, 2008), and SAM-T08 (Karchin, Cline & Karplus, 2004; Karchin et al., 2003; Karplus, 2009; Karplus & Hu, 2001; Karplus et al., 2001; Karplus et al., 2003; Karplus et al., 2005; Shackelford & Karplus, 2007), show that several of the FadD mutations cluster around a possible ATP/AMP entrance/exit channel (Fig. 4A and Fig. S3). All models have features similar to those of known adenylating enzymes as well as the acyl-CoA synthetase from Thermus thermophilus (Conti, Franks & Brick, 1996; Conti et al., 1997; Gulick, 2009; Gulick et al., 2003; Hisanaga et al., 2004; Hu et al., 2010). These include a small, globular C-terminal domain (white), a large, globular N-terminal domain (grey), and an active site (annotated by the alignment in Hisanaga et al. (2004)) situated between the two domains. Comparing these homology models to the structure of the Thermus thermophilus acyl-CoA synthetase shows that several of our FadD mutations cluster on a face of the protein from which ATP and AMP are proposed to enter and exit the active site (Hisanaga et al., 2004). Hisanaga et al. (2004) inferred that ATP binding precedes and enhances fatty acid binding, so enhancement of ATP binding would likely decrease the Km for the fatty acid. Given that our mutants fail to decrease Km, but do increase Vmax toward octanoate, we hypothesize that they could facilitate AMP exit from the active site by opening this face of the protein.


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

Ford TJ, Way JC - PeerJ (2015)

Rationally designed, site directed FadD mutants increase E. coli ΔfadR growth rate on octanoate when compared to wild-type FadD.(A) FadD homology model generated using The SWISS-MODEL Homology modeling server (Arnold et al., 2006; Benkert, Biasini & Schwede, 2011; Biasini et al., 2014) and the Thermus thermophilus structure as the template. The model was visualized in PyMOL with large N-terminal domain in gray, smaller C-terminal domain in white, and myristoyl-AMP (overlayed from the Thermus thermophillus structure) in yellow (myristoyl group) and magenta (AMP) (Hisanaga et al., 2004). Residues whose mutation results in increased growth rate on octanoate are color-coded according to the identity of the mutation (text below model, Y9H and V4F W5L are excluded from the model). (B) (i) Surface representation of the FadD homology model with residues mutated to glycine in (ii) shown in blue (mutations that decrease growth rate compared to wild-type) and red (mutations that increase growth rate compared to wild-type). (ii) Percent increase in exponential growth rate compared to wild-type FadD caused by mutating the residues on the X-axis to glycine. (C) (i) Cartoon representation of FadD homology model with residues mutated in (ii) in red. (ii) Percent increase in exponential growth rate compared to wild-type FadD caused by the FadD mutations depicted on the X-axis. n = 13–18, error bars indicate standard error in all cases, ** indicates growth rate significantly different from wild-type with p < 0.05 by two-sided students T-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493641&req=5

fig-4: Rationally designed, site directed FadD mutants increase E. coli ΔfadR growth rate on octanoate when compared to wild-type FadD.(A) FadD homology model generated using The SWISS-MODEL Homology modeling server (Arnold et al., 2006; Benkert, Biasini & Schwede, 2011; Biasini et al., 2014) and the Thermus thermophilus structure as the template. The model was visualized in PyMOL with large N-terminal domain in gray, smaller C-terminal domain in white, and myristoyl-AMP (overlayed from the Thermus thermophillus structure) in yellow (myristoyl group) and magenta (AMP) (Hisanaga et al., 2004). Residues whose mutation results in increased growth rate on octanoate are color-coded according to the identity of the mutation (text below model, Y9H and V4F W5L are excluded from the model). (B) (i) Surface representation of the FadD homology model with residues mutated to glycine in (ii) shown in blue (mutations that decrease growth rate compared to wild-type) and red (mutations that increase growth rate compared to wild-type). (ii) Percent increase in exponential growth rate compared to wild-type FadD caused by mutating the residues on the X-axis to glycine. (C) (i) Cartoon representation of FadD homology model with residues mutated in (ii) in red. (ii) Percent increase in exponential growth rate compared to wild-type FadD caused by the FadD mutations depicted on the X-axis. n = 13–18, error bars indicate standard error in all cases, ** indicates growth rate significantly different from wild-type with p < 0.05 by two-sided students T-test.
Mentions: FadD homology models generated using the SWISS-MODEL Homology modeling server (Arnold et al., 2006; Benkert, Biasini & Schwede, 2011; Biasini et al., 2014) and the Thermus thermophilus structure as the template, the I-TASSER server (Roy, Kucukural & Zhang, 2010; Yang et al., 2015; Zhang, 2008), and SAM-T08 (Karchin, Cline & Karplus, 2004; Karchin et al., 2003; Karplus, 2009; Karplus & Hu, 2001; Karplus et al., 2001; Karplus et al., 2003; Karplus et al., 2005; Shackelford & Karplus, 2007), show that several of the FadD mutations cluster around a possible ATP/AMP entrance/exit channel (Fig. 4A and Fig. S3). All models have features similar to those of known adenylating enzymes as well as the acyl-CoA synthetase from Thermus thermophilus (Conti, Franks & Brick, 1996; Conti et al., 1997; Gulick, 2009; Gulick et al., 2003; Hisanaga et al., 2004; Hu et al., 2010). These include a small, globular C-terminal domain (white), a large, globular N-terminal domain (grey), and an active site (annotated by the alignment in Hisanaga et al. (2004)) situated between the two domains. Comparing these homology models to the structure of the Thermus thermophilus acyl-CoA synthetase shows that several of our FadD mutations cluster on a face of the protein from which ATP and AMP are proposed to enter and exit the active site (Hisanaga et al., 2004). Hisanaga et al. (2004) inferred that ATP binding precedes and enhances fatty acid binding, so enhancement of ATP binding would likely decrease the Km for the fatty acid. Given that our mutants fail to decrease Km, but do increase Vmax toward octanoate, we hypothesize that they could facilitate AMP exit from the active site by opening this face of the protein.

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