A chemical genetic screen uncovers a small molecule enhancer of the N-acylethanolamine degrading enzyme, fatty acid amide hydrolase, in Arabidopsis Page: 4
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100Figure 2. Representative radiochromatograms of amidohydrolase activity of recombinant AtFAAH to
NAE 12:0. The reactions were initiated by the addition of 0.3 g of purified AtFAAH with: (a) DMSO (solvent
control); (b) 100 [M MDPD; and (c) 100 [M MDPD and boiled AtFAAH protein (negative control). The higher
FFA peak in assays with MDPD (arrow in the peak to the right in (b) indicates enhanced enzymatic activity.
enhanced by MDPD (Fig. 3a). In vitro amidohydrolase assays with ['4C]-NAE 12:0, - NAE 16:0, and - NAE 20:4
also were performed with the recombinant rat-FAAH enzyme with or without MDPD26. However, in our assay
conditions, we did not detect any significant enhancement of rat-FAAH activity by MDPD toward NAE 12:0 and
NAE 16:0. There was a slight but statistically insignificant increase in rat-FAAH activity toward NAE 20:4 in the
presence of MDPD (Fig. 3b).
To gain more insight into how MDPD modulates AtFAAH, we determined the kinetic parameters of
AtFAAH with and without MDPD. Consistent with previous studies, recombinant AtFAAH exhibited a typical
Michaelis-Menten behavior toward the NAE 12:0 substrate (Fig. 4)15,16. In the presence of MDPD, the apparent
Vm for AtFAAH was increased, but the apparent Km (27-28 M) of AtFAAH remained unchanged, indicating
that MDPD enhanced the rate of hydrolysis, but not the substrate affinity of AtFAAH. Indeed, MDPD increased
the catalytic efficiency of At FAAH by an estimated factor of-~3.4 (Fig. 4).
Given that MDPD could reverse the inhibitory effects of NAE 12:0 on seedling growth, we next tested whether
MDPD could also enhance endogenous amidohydrolase activity. We extracted total protein from 10 d old
wild-type seedlings and conducted amidohydrolase assays on protein extracts. In agreement with assays of the
recombinant AtFAAH protein, MDPD increased NAE hydrolytic activity in cell-free homogenates of seedlings
(Fig. 5a). The ability of MDPD to enhance in vivo amidohydrolase activity was also apparent from experiments
wherein the depletion of [1-'4C] -NAE 12:0 was followed in liquid cultures containing wild-type Arabidopsis
seedlings. Our results revealed that [1-'4C]_-NAE 12:0 was depleted by seedlings from the culture media but aRlI 17:411211 DOI: 10.1038/srep41121
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0ll'-"-A-.- .------.----- ---- -- I
AtFAAH + DMSO
NAE 12:0
FFA12:0
0 50 100 150
AtFAAH + MDPD
NAE 12:0
FFA12:0
0 50 100 150
AtFAAH boiled + MDPD
NAE 12:0
0 50 100 150
Position (mm)
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Khan, Bibi Rafeiza; Faure, Lionel; Chapman, Kent D. & Blancaflor, Elison B. A chemical genetic screen uncovers a small molecule enhancer of the N-acylethanolamine degrading enzyme, fatty acid amide hydrolase, in Arabidopsis, article, January 23, 2017; London, UK. (https://digital.library.unt.edu/ark:/67531/metadc967171/m1/4/: accessed April 25, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT College of Arts and Sciences.