Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana Page: 34,992
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Arabidopsis NAE Amidohydrolase
and purified, and enzyme assays were conducted as described previ-
ously (18) with a few modifications. Generally the enzyme source was
incubated with 100 M l"CINAE (20,000 dpm) in 50 mm BisTris buffer,
pH 9.0, for 30 min to survey for NAE amidohydrolase activity (18).
Enzyme activity was examined for time, temperature, protein, and
substrate concentration dependence. For enzyme characterization, re-
actions were initiated with 1 g of affinity-purified protein and incu-
bated at 30 C with shaking for 30 min. Assays of lysates of E. coli cells
expressing rat FAAH (WT-FAAH; Ref. 26) served as a comparison of
NAE amidohydrolase activity, whereas non-transformed cell lysates or
cell lysates with the Arabidopsis cDNA cloned in reverse orientation
with respect to the lacZ promoter served as negative controls for activ-
ity assays. Enzyme assays were terminated by the addition of boiling
isopropyl alcohol (70 *C), and lipids were extracted into chloroform.
Lipid products were separated by TLC, and the distribution of radioac-
tivity was evaluated by radiometric scanning (18). Activity was calcu-
lated based on the radiospecific activity of '4C-labeled substrate. A
general serine hydrolase inhibitor, phenylmethylsulfonyl fluoride
(PMSF), and an irreversible active site-directed FAAH inhibitor,
MAFP, were used to probe the sensitivity of recombinant Arabidopsis
NAE amidohydrolase activity. Inhibitors were added from stock solu-
tions dissolved in isopropyl alcohol for PMSF or Me2SO for MAFP, and
activity was adjusted for minimal solvent effects where necessary based
on assays in the presence of the appropriate amount of solvent alone.
Protein content was determined by Coomassie Blue dye binding using
bovine serum albumin as the protein standard (27).
RESULTS
Tentative Identification of Arabidopsis NAE Amidohydro-
lase-In animal tissues, fatty acid amide hydrolase (EC
3.5.1.4), a member of the amidase signature (AS) family (28,
29), hydrolyzes NAEs to produce FFA and ethanolamine (30). A
similar enzymatic activity was characterized previously in cot-
tonseed microsomes (18). Mammalian FAAH enzymes have a
conserved stretch of -130 amino acids (31) containing a Ser/
Ser/Lys catalytic triad (32). The predicted amidase structure
has a central conserved motif of GGSS(G/A/S)G (33), and a
somewhat longer stretch of amino acids G(GA)S(GS)(GS)GX(G-
SA)(GSAVY)X( LIVM)(GSA)X(6)(GSAT)X(GA)X(DE)X(GA)
XS(LIVM)RXP(GSAC) is present in all enzymes of the amidase
class (PS00457). Two serine residues at 217 and 241, highly
conserved in the AS sequence, were found essential for enzyma-
tic activity of recombinant rat FAAH (34). Mutation of either
one of the residues into alanine caused complete loss of activity
of the enzyme (34, 35). Also, mutation of serine 218 into alanine
caused marked loss of activity (35). Taking these conserved
residues in the AS consensus sequence into consideration'
several putative plant orthologues were identified computat-
ionally. BLAST searches2 in various data bases using the AS
consensus block embedded in rat FAAH (blocks.fhcrc.org)
identified one A. thaliana gene (At5g64440) that was selected
for further characterization (Fig. 1).
The structure and organization of the At5g64440 gene is
relatively complex with 21 exons including 5'-UTR (untrans-
lated region) and 3'-UTR (Fig. IA). The predicted gene is 4689
nucleotides in length and encodes a predicted protein of 607
amino acids with a molecular mass of 66.1 kDa. Based on the
presence of the conserved residues characteristic of the canon-
ical AS sequence, this gene seemed likely to encode an Arabi-
dopsis NAE amidohydrolase. To assess if this gene was ex-
pressed and to isolate a full-length cDNA for functional studies,
oligonucleotide primers were designed within the 5'- and 3'-
UTR, and a cDNA fragment was amplified by RT-PCR from
Arabidopsis leaf RNA (Fig. 1B). The RT-PCR product was
sequenced and found to be 99.9% identical with the correspond-
ing TC139316 annotated at TIGR. The protein domain predic-
tion tools, ProDom (20), identified six domain families in theArabidopsis protein, five of which were also found in rat FAAH
2 W. Gish, blast.wustl.edu.
(Fig. 1C). A single putative transmembrane segment was iden-
tified near the N terminus (TMHMM; Refs. 22 and 23) similar
to the predicted topological organization in rat FAAH.
Alignment of the deduced amino acid sequences from the
Arabidopsis NAE amidohydrolase cDNA and the rat FAAH
(28) showed only 18.5% identity over the entire length. Align-
ment within the AS sequence of 125 amino acids (Fig. 2A,
underlined residues) showed 37% identity with five residues
determined to be important for catalysis to be absolutely con-
served (Ref. 31; Lys-142, Ser-217, Ser-218, Ser-241, and Arg-
243, denoted by arrows; Fig. 2A). Comparison of a 47-amino
acid motif within the AS showed the Arabidopsis protein had
close to 60% identity with FAAHs from several mammalian
species (Fig. 2B). Organization of predicted secondary struc-
ture within this Arabidopsis and rat FAAH AS motif was
similar (Fig. 2C), and the structure of the rat enzyme has been
confirmed by x-ray crystallography (36). In addition, this pu-
tative A. thaliana NAE amidohydrolase and rat FAAH have
similar predicted molecular weights (-66 kDa), similar pre-
dicted topologies (single transmembrane segment near the N
terminus with the C terminus facing the cytosol, via TMHMM
transmembrane and topology predictor; Refs. 22 and 23), and
similar predicted subcellular locations (secretory pathway,
pSORT; Ref. 24). Although the Arabidopsis NAE amidohydro-
lase shared several domains with glutamyl-tRNA amidotrans-
ferases (Fig. 1C), these amino acid-modifying enzymes are lo-
calized to chloroplasts in higher plants, and there is no
indication of stromal targeting sequences at the N terminus of
the At5g64440 gene product.
Functional Identification of Arabidopsis NAE Amidohydro-
lase-The Arabidopsis putative NAE amidohydrolase was sub-
cloned into pTrcHis and pTrcHis2 for expression in E. coli of
N-terminal and C-terminal epitope- and polyhistidine-tagged
fusion proteins, respectively. E. coli lysates were surveyed for
expression of enzyme activity using ['4C]NAE 18:2 (radiola-
beled on the carbonyl carbon) as substrate. Representative
chromatograms shown in Fig. 3 indicate that, like the recom-
binant rat FAAH (expressed in the same vector), the recombi-
nant Arabidopsis protein effectively hydrolyzed [1-14C]NAE
18:2 to [1-14C]FFA 18:2. As a control, E. coli expressing the
Arabidopsis cDNA in reverse orientation showed no hydrolytic
activity (Fig. 3). In these preliminary experiments with crude
E. coli lysates, the Arabidopsis NAE amidohydrolase activity
was linear up to 60 min at protein amounts up to 50 g. The
enzyme was optimally active at alkaline pH (e.g. pH 8-9) and
was inactivated by treatment at 100 C for 10 min. The Arabi-
dopsis NAE amidohydrolase did not hydrolyze ceramide, nor
did ceramide influence NAE hydrolysis (not shown). The Ara-
bidopsis NAE amidohydrolase did not catalyze the reverse
reaction of NAE hydrolysis (formation of NAE) under any con-
ditions tested (not shown). Higher activity was reproducibly
recovered in cells expressing C-terminal fusions, compared
with cells expressing N-terminal fusions. Similar to reports for
the rat protein (26), the recombinant Arabidopsis NAE
amidohydrolase was mostly associated with E. coli membranes.
Affinity Purification of Recombinant Enzyme-The Arabi-
dopsis NAE amidohydrolase, expressed as a C-terminal fusion
protein, was solubilized in DDM and subjected to native Ni2+-
affinity purification, SDS-PAGE, Western blot analyses, and
enzyme activity assays (Fig. 4). A protein of -70 kDa was
enriched under native conditions by Ni2+-affinity purification
and was detected by the c-Myc antibody (Fig. 4, A and B,
arrows, Rec. protein lanes). Likewise, NAE amidohydrolaseactivity was enriched in this native affinity-purified protein
fraction (Fig. 4C) by -375-fold, relative to the DDM-solubilized
supernatant (Supt) fraction. More stringent denaturing condi-34992
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Shrestha, Rhidaya; Dixon, R. A. & Chapman, Kent D. Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana, article, June 24, 2003; [Rockville, Maryland]. (https://digital.library.unt.edu/ark:/67531/metadc485928/m1/3/: accessed July 18, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT College of Arts and Sciences.