Difference between revisions of "Part:BBa K654059"
 
(4 intermediate revisions by 2 users not shown)
Line 13: Line 13:
  
  
−
<!-- Add more about the biology of this part here
+
==FadD Information Contribution by team William_and_Mary 2020==
−
===Usage and Biology===
+
  
−
<!-- -->
+
===Protein Function & Overview===
−
<span class='h3bb'>Sequence and Features</span>
+
−
<partinfo>BBa_K654059 SequenceAndFeatures</partinfo>
+
  
 +
FadD is a soluble fatty acyl CoA synthetase endogenous to Escherichia coli. It is classified as an AMP-forming fatty acid CoA ligase, meaning that it combines fatty acids with Coenzyme A molecules in a reaction that is powered by converting ATP to AMP. FadD activates both medium and long chain fatty acids into fatty acyl CoA thioesters, which are substrates for beta oxidation, phospholipid biosynthesis, and cellular signalling. Beta oxidation is the pathway that degrades fatty acids, which can be regulated by fatty acyl CoA thioesters. (Yoo, 2001)
  
−
<!-- Uncomment this to enable Functional Parameter display
+
FadD can be found within the cell by the plasma membrane, where it is non-integrally associated. Its activity is enhanced by the membrane lipids or detergents nearby. The protein has a molecular weight of 62,028 Daltons, though it forms a dimer with a molecular weight of about 120,000. It is contained within a 2.2 kilobase fragment of the E. coli genome, as part of a fatty acid degradative regulon along with fadBA, fadE, and fadL, all under control of repressor FadR. Transcription starts 60 base pairs upstream of the translation start. After the translational stop is a GC-rich inverted repeat and a 8T transcriptional terminator. Two FadR operator sites are found at -13 to -29 and -99 to -115. A rare UUG codon at translation initiation may downregulate expression. (Black, 1992)
−
===Functional Parameters===
+
−
<partinfo>BBa_K654059 parameters</partinfo>
+
−
<!-- -->
+
  
−
== Characterized by BNU-China 2019 ==
+
After long chain fatty acyl-CoA are sequestered inside the cell by vectorial thioesterification with FadD, they can bind repressor protein FadR. This binding to FadR causes it to dissociate from operator sites on the fatty acid degradative (Fad) regulon, relieving the repression on the regulon transcription such that Fad genes can be expressed. (Yoo, 2001)
  
−
In order to have our engineered microbe consume the extra in-taken fat, we overexpress fadD gene derived from E. coli K-12 DH5alpha genome in our engineered intestinal microbe to promote degradation of higher fatty acids which would otherwise be assimilated by human body. The catalysate fatty acyl-CoA also enhances the general fatty acids degradation by relieving the overall inhibiting effect of regulatory factor fadR towards β-oxidation. [1]
+
===Chemical Reactions, Substrate Specificity, and Kinetics===
  
−
Considering that sodium oleate has a generally steady and relatively high content in most kinds of fat, we select it to test relative general consumption of higher fatty acids.
+
Mechanistic equation: FA + ATP = FA-AMP (needs Mg2+), FA-AMP + CoASH = FA-SCoA. FadD activates fatty acids by converting their carboxyl group into an acyl-CoA thioester, which is a stronger electrophile. Fatty acids enter the FadD active site from the membrane through a narrow channel that faces the inner membrane while ATP enters through a distinct large channel. Binding these molecules causes FadD to undergo ligand-induced conformational changes. The molecules then form an AMP-FA intermediate, which the flexible C-terminal clamps in order to position the intermediate and prevent its escape. CoA, the final substrate, binds to FadD after the fatty acid and ATP. CoA enters the FadD active site via a third channel and attacks the new bond, generating FA-CoA and AMP. Supposedly, when CoA bonds to a long chain fatty acid, AMP is pushed from the active site by the LCFA-CoA product, but this push is less pronounced with MCFA. (Ford, 2015)
  
−
We take E. coli introduced with a vector with the same backbone as control group. Compared to it, the experimental group shows a significant increase in fatty acids consumption upon induction. As is shown in Fig. 1, the experimental group consumes more than twice as much sodium oleate as the control group within 2 and 4 hours, indicating enhancement of β-oxidation consume an extra amount of higher fatty acids is achieved by overexpressing fadD gene.
+
FadD belongs to a class of adenylate forming enzymes, whose fatty acid tunnel length determines substrate specificity by accommodating a long hydrophobic tail. FadD has broad chain length specificity with a Vmax ranging from 2632 nmol/min/mg protein for C12 to 135 for C6. However, its maximal activity is reserved for fatty acids with carbon numbers ranging between 12 and 18, activating both mono- and poly-unsaturated fatty acids. The thioesters synthesized are destined for degradation or phospholipid incorporation. FadD has lower activity on medium chain fatty acids with 6 to 12 carbons. Downstream beta oxidation enzymes also have poor activity on MCFA. (Black, 1992)
  
 +
===Interactions with other Proteins: Repression by FadR and Cleavage by OmpT===
  
−
<font size="4"><b>Experimental approach</b></font>
+
The FadD gene contains two FadR binding sites. The first operator (located from -13 to -29) has 12/17 consensus and the second operator (-115 to -98) has 9/17 consensus. FadD’s operator sites have Keq equal to 10-9 M and 10-8 M, compared to Keq equal to 3 x 10-10 M for FadB’s operator site, the strongest binding site for FadR in E. coli. Long chain fatty acyl CoA prevents FadR from having affinity for operator 1, which would otherwise turn off fadD transcription. (Black, 1992)
  
−
1.Transform the plasmids into E. coli DH5α competent cells.
+
FadD is a substrate for OmpT in vitro, which cleaves the 62 kDa protein into a 42.97 kDa C-terminal fragment and a 19.39 kDa N-terminal fragment. The cleavage site between residues lysine 172 and arginine 173 is a linker domain that connects the ATP and LCFA binding C-terminal to the N-terminal. The outer membrane serine protease OmpT has dibasic residue specificity with its active site on the cell surface. (Yoo, 2001)
  
−
2.A strain containing a vector with same backbone is used as control. Experimental groups and control groups are cultured in LB-ampicillin (50 ng/µl) medium overnight before being diluted with equal amount of LB-ampicillin (50 ng/µl) medium containing 400 mM sodium oleate, making the final concentration of oleate 200 mM.
+
Cleavage did not affect binding ability and the fragments remained associated afterwards. OmpT cleaving FadD results in FadD’s new Km and Vmax values twice as high, but catalytic efficiency remains similar. Enzymatic activity is retained because the hydrocarbon chain of fatty acids interacts with the 43 kDa fragment and the AMP binding signature motif binds nucleotides in the same 43 kDa fragment. Adjacent to where the hydrocarbon binds, consensus 25 amino acid fatty acyl CoA signature motif is involved in substrate specificity and binding. While all the binding domains are within the 43 kDa C-terminal fragment, it is unstable alone. The N-terminal remains associated because it is required for structural stability. (Yoo, 2001)
  
−
3.Both groups are induced with 5 mM IPTG and sampled at 0 hr, 2 hr and 4 hr. Centrifuge samples and take the supernatant.
+
When the substrate oleate was present, it inhibited the protease OmpT by changing FadD conformation to protect the cleavage site. Adding ATP allowed FadD to reset its conformation, so the site was exposed again. Sensitivity to proteolysis is correlated with increased mobility and flexibility, so changing the conformation of the flexible hinge by binding LCFA or ATP or interacting with detergent can alter the accessibility of the OmpT cleavage site. (Yoo, 2001)
  
−
4.Measure the fatty acids concentration through enzyme linked immunosorbent assay (Shuangying FFA ELISA kit).
+
===Mutations===
 +
Mutations clustered on the face of FadD where AMP exits enhance activity by aiding product exit. Growth rate increased with a wider AMP exit channel from the active site, although some of the mutants had decreased activity on long chain fatty acids. FadD mutations that increase the rate of reaction do not enhance affinity for MCFA. ATP binding precedes and enhances fatty acid binding, so mutants that increase activity facilitate AMP exit and ATP entry from the active site. Removing amino acid side chains surrounding the ATP/AMP channel destabilizes the closed conformation. Mutation eases transition to open state by enhancing AMP exit. These mutations affect the structure of the AMP exit channel or interact with the C-terminal domain. (Ford, 2015)
  
−
5.Calculate and compare the sodium oleate consumption of experimental group and control group.
+
===Bioengineering===
 +
Synthetic biologists have engineered E. coli to be a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. When paired with fadE (acyl-CoA dehydrogenase) disruption and tesA (acetyl-CoA thioesterase) overexpression, overexpression of fadD has enhanced the synthesis of fatty alcohols, olefins, and free fatty acids. For all these biofuels, other steps were involved to maximize production: fatty alcohols required accABCD (acetyl-CoA carboxylase) and acrI (acyl-CoA reductase from A. baylyi) co-overexpression, olefins required oleABCD (beta-ketoacyl-ACP synthase from S. maltophilia) overexpression, and FFA required fatty acid synthase (fabH, fabD, fabG, fabF) and accABCD overexpression. (Clomburg, 2010) (Colin, 2011)
  
−
6.Three repicas are tested in each group.
+
===Related proteins===
 +
Examining fadD’s amino acid sequence alongside rat and yeast acyl CoA synthetases reveals a high degree of similarity in the carboxyl end (353-455), which might be an ATP binding domain because it is also shared with firefly luciferase. High similarity to the other acyl CoA synthetases at a central location (200-273) might indicate a fatty acid or Coenzyme A binding domain. (Black, 1992)
  
 +
There is a second acyl-CoA synthetase in E. coli: FadK. FadK has a higher relative rate on medium chain fatty acids than long chain, but lower absolute activity in comparison to FadD. FadK is only expressed under anaerobic conditions. An unidentified H+/LCFA cotransporter might be present in the inner membrane to interact directly with acyl CoA synthetase. (Ford, 2015)
  
−
<font size="4"><b>Reference</b></font>
 
  
−
[1] Zhang Han‐Xing. Screening of PoIyhydroxyalkanoates producing bacteria and its expression and metabolic mechanism in E. coli engineered bacteria: [D]. Jinan: Shandong University, 2006
+
===References===
 +
 
 +
Black, P. N., DiRusso, C. C., Metzger, A. K., & Heimert, T. L. (1992). Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. The Journal of biological chemistry, 267(35), 25513–25520.
 +
 
 +
Yoo, J. H., Cheng, O. H., & Gerber, G. E. (2001). Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT. The Biochemical journal, 360(Pt 3), 699–706. https://doi.org/10.1042/0264-6021:3600699
 +
 
 +
Ford, T. J., & Way, J. C. (2015). Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids. PeerJ, 3, e1040. https://doi.org/10.7717/peerj.1040
 +
 
 +
Clomburg, J. M., & Gonzalez, R. (2010). Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology. Applied microbiology and biotechnology, 86(2), 419–434. https://doi.org/10.1007/s00253-010-2446-1
 +
 
 +
Colin, V. L., Rodríguez, A., & Cristóbal, H. A. (2011). The role of synthetic biology in the design of microbial cell factories for biofuel production. Journal of biomedicine & biotechnology, 2011, 601834. https://doi.org/10.1155/2011/601834
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:20, 23 October 2020

Fatty Acyl-CoA Synthetase (Ligase) (fadD from E. coli) with 8-His Tag

FadD Fatty Acyl-CoA Synthetase (Ligase) from E. coli. Contains a 8-Histidine tag immediately before the stop codon to allow gel identification and extraction. The FadD Fatty Acyl-CoA Synthetase (Ligase) converts fatty acids into fatty acyl-CoA using ATP.


2011 Utah State FattyAcylCoASynthetase.png


Fatty Acyl-CoA Synthetase is used in conjunction with Thioesterase to greatly increase the concentration of Fatty Acyl-CoA present in the cell, to increase the amount of bioproducts produced. It is used to produce Fatty Alcohols and Wax Esters in the 2011 Utah_State iGEM project.


2011 Utah State Pathway 2.png


FadD Information Contribution by team William_and_Mary 2020

Protein Function & Overview

FadD is a soluble fatty acyl CoA synthetase endogenous to Escherichia coli. It is classified as an AMP-forming fatty acid CoA ligase, meaning that it combines fatty acids with Coenzyme A molecules in a reaction that is powered by converting ATP to AMP. FadD activates both medium and long chain fatty acids into fatty acyl CoA thioesters, which are substrates for beta oxidation, phospholipid biosynthesis, and cellular signalling. Beta oxidation is the pathway that degrades fatty acids, which can be regulated by fatty acyl CoA thioesters. (Yoo, 2001)

FadD can be found within the cell by the plasma membrane, where it is non-integrally associated. Its activity is enhanced by the membrane lipids or detergents nearby. The protein has a molecular weight of 62,028 Daltons, though it forms a dimer with a molecular weight of about 120,000. It is contained within a 2.2 kilobase fragment of the E. coli genome, as part of a fatty acid degradative regulon along with fadBA, fadE, and fadL, all under control of repressor FadR. Transcription starts 60 base pairs upstream of the translation start. After the translational stop is a GC-rich inverted repeat and a 8T transcriptional terminator. Two FadR operator sites are found at -13 to -29 and -99 to -115. A rare UUG codon at translation initiation may downregulate expression. (Black, 1992)

After long chain fatty acyl-CoA are sequestered inside the cell by vectorial thioesterification with FadD, they can bind repressor protein FadR. This binding to FadR causes it to dissociate from operator sites on the fatty acid degradative (Fad) regulon, relieving the repression on the regulon transcription such that Fad genes can be expressed. (Yoo, 2001)

Chemical Reactions, Substrate Specificity, and Kinetics

Mechanistic equation: FA + ATP = FA-AMP (needs Mg2+), FA-AMP + CoASH = FA-SCoA. FadD activates fatty acids by converting their carboxyl group into an acyl-CoA thioester, which is a stronger electrophile. Fatty acids enter the FadD active site from the membrane through a narrow channel that faces the inner membrane while ATP enters through a distinct large channel. Binding these molecules causes FadD to undergo ligand-induced conformational changes. The molecules then form an AMP-FA intermediate, which the flexible C-terminal clamps in order to position the intermediate and prevent its escape. CoA, the final substrate, binds to FadD after the fatty acid and ATP. CoA enters the FadD active site via a third channel and attacks the new bond, generating FA-CoA and AMP. Supposedly, when CoA bonds to a long chain fatty acid, AMP is pushed from the active site by the LCFA-CoA product, but this push is less pronounced with MCFA. (Ford, 2015)

FadD belongs to a class of adenylate forming enzymes, whose fatty acid tunnel length determines substrate specificity by accommodating a long hydrophobic tail. FadD has broad chain length specificity with a Vmax ranging from 2632 nmol/min/mg protein for C12 to 135 for C6. However, its maximal activity is reserved for fatty acids with carbon numbers ranging between 12 and 18, activating both mono- and poly-unsaturated fatty acids. The thioesters synthesized are destined for degradation or phospholipid incorporation. FadD has lower activity on medium chain fatty acids with 6 to 12 carbons. Downstream beta oxidation enzymes also have poor activity on MCFA. (Black, 1992)

Interactions with other Proteins: Repression by FadR and Cleavage by OmpT

The FadD gene contains two FadR binding sites. The first operator (located from -13 to -29) has 12/17 consensus and the second operator (-115 to -98) has 9/17 consensus. FadD’s operator sites have Keq equal to 10-9 M and 10-8 M, compared to Keq equal to 3 x 10-10 M for FadB’s operator site, the strongest binding site for FadR in E. coli. Long chain fatty acyl CoA prevents FadR from having affinity for operator 1, which would otherwise turn off fadD transcription. (Black, 1992)

FadD is a substrate for OmpT in vitro, which cleaves the 62 kDa protein into a 42.97 kDa C-terminal fragment and a 19.39 kDa N-terminal fragment. The cleavage site between residues lysine 172 and arginine 173 is a linker domain that connects the ATP and LCFA binding C-terminal to the N-terminal. The outer membrane serine protease OmpT has dibasic residue specificity with its active site on the cell surface. (Yoo, 2001)

Cleavage did not affect binding ability and the fragments remained associated afterwards. OmpT cleaving FadD results in FadD’s new Km and Vmax values twice as high, but catalytic efficiency remains similar. Enzymatic activity is retained because the hydrocarbon chain of fatty acids interacts with the 43 kDa fragment and the AMP binding signature motif binds nucleotides in the same 43 kDa fragment. Adjacent to where the hydrocarbon binds, consensus 25 amino acid fatty acyl CoA signature motif is involved in substrate specificity and binding. While all the binding domains are within the 43 kDa C-terminal fragment, it is unstable alone. The N-terminal remains associated because it is required for structural stability. (Yoo, 2001)

When the substrate oleate was present, it inhibited the protease OmpT by changing FadD conformation to protect the cleavage site. Adding ATP allowed FadD to reset its conformation, so the site was exposed again. Sensitivity to proteolysis is correlated with increased mobility and flexibility, so changing the conformation of the flexible hinge by binding LCFA or ATP or interacting with detergent can alter the accessibility of the OmpT cleavage site. (Yoo, 2001)

Mutations

Mutations clustered on the face of FadD where AMP exits enhance activity by aiding product exit. Growth rate increased with a wider AMP exit channel from the active site, although some of the mutants had decreased activity on long chain fatty acids. FadD mutations that increase the rate of reaction do not enhance affinity for MCFA. ATP binding precedes and enhances fatty acid binding, so mutants that increase activity facilitate AMP exit and ATP entry from the active site. Removing amino acid side chains surrounding the ATP/AMP channel destabilizes the closed conformation. Mutation eases transition to open state by enhancing AMP exit. These mutations affect the structure of the AMP exit channel or interact with the C-terminal domain. (Ford, 2015)

Bioengineering

Synthetic biologists have engineered E. coli to be a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. When paired with fadE (acyl-CoA dehydrogenase) disruption and tesA (acetyl-CoA thioesterase) overexpression, overexpression of fadD has enhanced the synthesis of fatty alcohols, olefins, and free fatty acids. For all these biofuels, other steps were involved to maximize production: fatty alcohols required accABCD (acetyl-CoA carboxylase) and acrI (acyl-CoA reductase from A. baylyi) co-overexpression, olefins required oleABCD (beta-ketoacyl-ACP synthase from S. maltophilia) overexpression, and FFA required fatty acid synthase (fabH, fabD, fabG, fabF) and accABCD overexpression. (Clomburg, 2010) (Colin, 2011)

Related proteins

Examining fadD’s amino acid sequence alongside rat and yeast acyl CoA synthetases reveals a high degree of similarity in the carboxyl end (353-455), which might be an ATP binding domain because it is also shared with firefly luciferase. High similarity to the other acyl CoA synthetases at a central location (200-273) might indicate a fatty acid or Coenzyme A binding domain. (Black, 1992)

There is a second acyl-CoA synthetase in E. coli: FadK. FadK has a higher relative rate on medium chain fatty acids than long chain, but lower absolute activity in comparison to FadD. FadK is only expressed under anaerobic conditions. An unidentified H+/LCFA cotransporter might be present in the inner membrane to interact directly with acyl CoA synthetase. (Ford, 2015)


References

Black, P. N., DiRusso, C. C., Metzger, A. K., & Heimert, T. L. (1992). Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. The Journal of biological chemistry, 267(35), 25513–25520.

Yoo, J. H., Cheng, O. H., & Gerber, G. E. (2001). Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT. The Biochemical journal, 360(Pt 3), 699–706. https://doi.org/10.1042/0264-6021:3600699

Ford, T. J., & Way, J. C. (2015). Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids. PeerJ, 3, e1040. https://doi.org/10.7717/peerj.1040

Clomburg, J. M., & Gonzalez, R. (2010). Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology. Applied microbiology and biotechnology, 86(2), 419–434. https://doi.org/10.1007/s00253-010-2446-1

Colin, V. L., Rodríguez, A., & Cristóbal, H. A. (2011). The role of synthetic biology in the design of microbial cell factories for biofuel production. Journal of biomedicine & biotechnology, 2011, 601834. https://doi.org/10.1155/2011/601834


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]