Part:BBa_K1712000

E. coli FABI

This sequence is of the E. coli FABI that encodes for the organism's enoyl acyl-carrier-protein reductase in type 2 fatty acid synthesis. The enzyme this encodes for uses NADH and crotonyl COA as substrates.

Fatty acid Biosynthetic Pathway

Triclosan inhibits type 2 fatty acid synthesis (FASII), an essential pathway in the Bacterial and Eukaryotic domains by interacting directly with the enoyl acyl carrier protein reductase (FabI)[3]. Evidence that we could use the enzyme to detect triclosan came from binding studies and crystallographic data initially came from Heath et al. They showed that triclosan binding increases the enzyme’s afinity for NAD+ and triclosan’s role as an effective inhibitor is due to the formation of a stable ternary complex between fabI, triclosan, and NAD+ [4].

Usage and Biology

The basis of our biosensor, therefore, is to use triclosan’s mechanism of action as an inhibitor of enoyl acyl carrier protein reductase (fabI) in order to detect it. To screen a representative subset of fabI’s from all available domains of life, we mined the literature and found every characterized FabI with published inhibition data. We found all living organisms except for the Archaea, who synthesize lipids based on isoprenoides, have a fabI gene [5].

Protein purity assessed through SDS-PAGE gels shown below: Enzyme Expression Assay:

Unlike the other enzymes of the FASII pathway, it’s very important to note there is considerable diversity in the structure of FabI’s from different organisms[5]. AND, not all of them have the same level of sensitivity towards triclosan[23]. This is why we needed to screen a panel of enzymes in order to find the enzyme most sensitive towards triclosan with a nanomolar to inhibition constant. (See above for why we wanted to see nanomolar inhibition)

Enzyme Activity Assay:

Enzyme activity was measured spectrophotometrically through the decrease in NADH absorbance over time on the native substrate analog crotonyl CoA. Activity is defined as the change in optical density (absorbance) per minute. Activity is normalized by dividing activity by the microgram of enzyme used for the assay. Each enzyme was assayed with 100 uM NADH and 100 uM crotonyl-CoA. Negative control was 100 uM NADH, 100 uM crotonyl-CoA, no enzyme. Observed enzyme activities were subtracted from negative control and plotted on a log scale. Two biological replicates of each enzyme was used.

Triclosan Inhibition Screening

Since Chalew et al showed the levels of triclosan leaving Waste Water Treatment Plants (WWTPs) was up to 9 nanomolar [18], we wanted to measure enzyme inhibition using a nanomolar level of triclosan. Under our conditions, however, not all of the fabI enzymes had measurable activity with a nanomolar amount of enzyme, and in order to see inhibition using a nanomolar amount of triclosan we needed to use a nanomolar amount of enzyme.

Enzyme Inhibition Assay:

Triclosan inhibition was measured by running our standard enzyme activity assay with no triclosan and 1 nM triclosan. Negative control was 100 uM NADH, 100 uM crotonyl CoA, no enzyme, no triclosan. Observed enzyme activities were subtracted from negative control activities. Percent inhibition was calculated by:

( (uninhibited activity - inhibited activity) / uninhibited activity ) * 100

The enzyme concentrations ranged from 1.9 - 3.3 nM using two biological replicates of each enzyme. Nanomolar inhibition from P. falciparum has been previously reported [8], but we are the first to show triclosan inhibition using A. protothecoides Fabi.

Interestingly, with P. tricornutum, a marine diatom, which have been shown to be sensitive towards triclosan [29] we were unable to see triclosan inhibition. However, nanomolar inhibition was also not observed with H. influenzae, and E.coli, even though previously reported [6][7][10].

Characterization and Codon Optimization of Previous Part

We codon optimized and characterized a previous part: https://parts.igem.org/Part:BBa_K771303 This was done for growth and expression in E. coli with the pET29b+ plasmid. The protein sequences are the same, but the nucleotides were codon optimized, see below for the alignment.

BBa_K771303 and BBa_K1712000 Nucleotide Alignment:

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]

References

[3] McMurry, Laura M., Margret Oethinger, and Stuart B. Levy. "Triclosan Targets Lipid Synthesis." Nature 394 (1998): 531-32. Web. [4] Heath, R. J. , Yu, Y.-T. , Shapiro, M. A. , Olson, E. & Rock, C. O. J. Biol. Chem. 273, 30316–30320 (1998) [5] RP, Massengo-Tiassé, and Cronan JE. "Diversity in Enoyl-acyl Carrier Protein Reductases." Cell Mol Life Sci. (May 2009): n. pag. Web. [6] RJ, Heath, Rubin JR, Holland DR, Zhang E, Snow ME, and Rock CO. "Mechanism of Triclosan Inhibition of Bacterial Fatty Acid Synthesis." J Biol Chem (April 1999): n. pag. Web. [7] Ward, Walter. "Kinetic and Structural Characteristics of the Inhibition of Enoyl (acyl Carrier Protein) Reductase by Triclosan." Biochemistry (1999 Sep 21): n. pag. Web. [8] Kapoor, Mili. "Slow-tight-binding Inhibition of Enoyl-acyl Carrier Protein Reductase from Plasmodium Falciparum by Triclosan." Biochem (2004 August 1): n. pag. Web. [10] Marcinkeviciene, J.et al, (2001). "Enoyl-ACP Reductase (FabI) of Haemophilus influenzae: Steady-State Kinetic Mechanism and Inhibition by Triclosan and Hexachlorophene." Archives of Biochemistry and Biophysics 390(1): 101-108. [18] Chalew T. E., Halden R. U. (2009). Environmental exposure of aquatic and terrestrial biota to triclosan and triclocarban. J. Am. Water Works Assoc. 45, 4–13. 10.1111/j.1752-1688.2008.00284.x [23]. Mol. Biol. (2004) 343, 147–155 doi:10.1016/j.jmb.2004.08.033 [29] Triclosan causes toxic effects to algae in marine biofilms, but does not inhibit the metabolic activity of marine biofilm bacteria Marine Pollution Bulletin 84 (2014) 208–212