BACKGROUND
anandamide  <cit>  is a mammalian endogenous lipid that binds cannabinoid receptors which are mainly present in the central nervous system and immune cells.
anandamide was identified in  <dig> and named after the sanskrit word ananda, meaning bliss or delight.
anandamide acts as an agonist for the central cannabinoid receptor  and is therefore referred to as cannabinoid.
it mimics pharmacological effects of Δ9tetrahydrocannabinol, an active ingredient of marijuana  <cit> .
action of anandamide is terminated by the enzyme fatty acid amide hydrolase   <cit> .
faah was originally identified in  <dig> from rat liver plasma membrane and later faah homologs were identified from other sources including human, porcine, and arabidopsis.
faah belongs to a large group of proteins containing a conserved amidase signature motif  <cit> .
faah can also hydrolyze, in addition to anandamide, other fatty acid derivatives like n-oleoylethanolamine and n-palmitoylethanolamine collectively referred as n-acylethanolamines   <cit> .
studies on mammalian faah have provided more information on naes role in regulating various physiological functions like sleep and pain  <cit> .
recent studies on naes reveal further biological roles in appetite suppression, vasodilatation, cardiac function and inflammation  <cit> .
therefore any faah inhibitors which intervene in nae’s bioactivity promise to be a novel class of therapeutics and much drug discovery research is being actively pursued in this regard  <cit> .
anandamide is yet to be found in dictyostelium, but its precursor n-acylphosphatidylethanolamine  has previously been identified  <cit> .
in mammalian cells anandamide is believed to originate from hydrolysis of nape by phospholipase d .
in dictyostelium, a pld homolog pldb was identified and proposed to have a similar function  <cit> .
identification of faah suggests that regulation of nae signalling could occur in dictyostelium and thus dictyostelium could be utilised as a simple eukaryotic model to study nae functions in parallel with mammalian systems.
dictyostelium has been used to study cell motility, chemotaxis, cell differentiation and morphogenesis enabling significant contributions to an understanding of similar processes in mammalian systems.
recently dictyostelium has been used as a system to study the mechanism of action for mood stabilizing drugs like lithium and valproic acid  <cit> .
therefore the identification of any new drug target enzyme such as faah or any drug processing mechanisms in dictyostelium suggests further potential for the use of dictyostelium in human biomedical research.
dictyostelium offers an attractive system to study such processes by gene manipulation studies because of its small  <dig> mbp haploid genome harbouring many homologous genes found in higher eukaryotes  <cit> .
RESULTS
amino acid sequence analysis
a putative faah in dictyostelium was identified by a bioinformatics approach searching for a human faah homolog in the dictyostelium genome.
dictyostelium dna sequence ddb_g <dig>   containing coding sequences for characteristic amidase signature motifs  <cit>  was identified and found to be located on chromosome  <dig> in the annotated dictyostelium genome data base.
will be referred to as dictyostelium faah as the protein’s amino acid sequence analysis and other experimental results confirm its function to be similar to mammalian faah.
the calculated molecular weight of dictyostelium faah is 70 kda and domain architecture analysis  reveals the presence of an amidase domain composed of a characteristic amidase signature  sequence .
the consensus amidase signature sequence has a conserved gssg  motif shared among many proteins in the amidase class including glutamyl-t-rna amidotransferase subunit a of methanococcus jannaschii and faah from human, porcine, rat, arabidopsis and dictyostelium.
faah from human, porcine and rat are composed of  <dig> amino acids and faah from dictyostelium and arabidopsis contain  <dig> and  <dig> amino acids, respectively.
faah full length protein amino acid sequence from dictyostelium lacks significant identity when compared to faah from human , porcine , rat , and arabidopsis  , but identity across the amidase signature sequence increased to 40%, 38%, 38%, and 50%, for the human, procine, rat, and arabidopsis faah homologs.
the serine residues at  <dig> and  <dig> found to be essential for rat faah activity  <cit>  were also conserved in as sequence of dictyostelium faah.
other catalytically important residues lys <dig>  ser <dig> and arg <dig> found in rat were also conserved in dictyostelium.
recombinant enzyme expression and affinity purification of faah in dictyostelium and e. coli
faah was expressed in dictyostelium as an n-terminal his tag fusion protein.
faah was found to be predominantly a membrane associated protein and to improve yield of the purified protein, a  <dig> % concentration of triton x- <dig> was used in lysis buffer to solubilise membrane fractions.
cells expressing recombinant his-faah protein  were solubilised in lysis buffer and subjected to ni-nta affinity chromatography separation.
purified protein obtained was analyzed by coomassie staining  and western blotting analysis  using anti-his antibody  and anti-faah polyclonal antibody  respectively.
initial attempts to express faah as a his tag fusion protein in e.coli were not successful, as both n-terminal his and c-terminal his fusions to faah were unstable and only a small amount of the protein was made and this was only found in inclusion bodies.
alternatively, in order to simplify large scale recombinant protein production, faah was expressed and purified as a recombinant maltose binding protein  fusion protein from e.coli .
recombinant faah when expressed as n-terminal mbp fusion protein  in e.coli produced a higher yield of soluble recombinant protein.
recombinant faah when produced in either dictyostelium or e.coli migrated on sds-polyacrylamide gels, consistent with no significant post-translation modification.
functional identification of dictyostelium faah
dictyostelium recombinant faah expressed as n-terminal his tagged fusion protein  in dictyostelium and as n-terminal mbp tagged fusion protein  in e.coli hydrolyzed anandamide to free arachidonic acid and ethanolamine as determined by ce-es-ms .
dictyostelium faah was also capable of hydrolyzing synthetic p-nitroanilide substrates arachidonoyl p-nitroaniline  and decanoyl p-nitroaniline  which were further used in kinetics studies.
catalytic properties
recombinant his-faah purified from dictyostelium was analyzed for fatty acid amide hydrolase activity by measuring the rate of hydrolysis of p-nitroanilide substrates apna  and dpna  , which were previously used to characterize binding and catalytic specificities of mammalian faah enzymes  <cit> .
dictyostelium faah exhibited michaelis-menten kinetics on these substrates.
specific constant kcat/km values  observed for apna having long chain unsaturated fatty acid  were slightly higher when compared to dpna , which may indicate the enzyme’s preference for longer unsaturated acyl chains.
similar observations were made with mammalian faah where the enzyme showed a  <dig> fold preference for anandamide versus n-palmitoylethanolamine  <cit> .
the kcat values of his-faah towards apna and dpna when compared with rat faah were about  <dig> and  <dig> times less, respectively.
purified recombinant faah enzymes from both dictyostelium and e.coli exhibited ph optima at  <dig>  which were similar to the mammalian faah enzymes characterized to have a ph optimum from  <dig> to  <dig>  compounds that inhibit enzymatic activity via different mechanisms, phenylmethylsulfonyl fluoride , ly <dig> and methyl arachidonoyl fluorophosphonate  were tested on dictyostelium faah in order to monitor changes in activity.
non-specific irreversible serine protease inhibitor pmsf was modestly effective and inhibited about 58% at 5 mm .
ly <dig>  a serine hydrolase inhibitor which inactivates faah by covalent binding, was also modestly effective against dictyostelium faah and inhibited about 62% at  <dig>  mm .
irreversible active site targeted inhibitor mafp had potent inhibition against dictyostelium faah and inhibited about 63% at  <dig> μm .
identification of faah in dictyostelium
the production of faah protein in dictyostelium was confirmed at the protein level.
dictyostelium anti-faah polyclonal antibodies raised in rabbits  were used to detect faah production during dictyostelium development.
to trace the in vivo faah protein production profile, wild type dictyostelium cells allowed to develop on phosphate agar plates at different stages of development from independent single cell stage through multi-cellular fruiting body, were harvested.
total proteins isolated from the harvested cells were analyzed for faah expression by western blotting using anti faah polyclonal antiserum.
faah was identified as a predicted 70 kda protein expressed at constant levels throughout all the different stages of dictyostelium development suggesting an essential role for faah throughout development.
however, expression levels of in vivo faah protein in dictyostelium wild type cells were very low and several attempts to study protein localization by cell fractionation and western blotting were not successful.
the inability to detect endogenous faah protein in the fractionation experiments may be due to very low level of protein expression or due to protein getting degraded during the process of fractionation.
therefore, ax3faah cells were used in cell fractionation studies.
cells grown in liquid nutrient were harvested and fractionated into membrane and cytosol fractions by differential centrifugation.
at  <dig> xg, faah was distributed in both pellet and supernatant fractions  indicating that faah may be a plasma membrane associated protein.
at  <dig> xg, faah was predominantly present in pellet fraction further indicating that faah may be associated with other intra cellular membrane bound organelles.
the small quantities of faah in the supernatant after this spin strongly suggest a predominantly membrane associated protein and is further supported by increased yields of his-faah when detergents such as triton x- <dig> are added.
unlike other mammalian faahs, dictyostelium faah does not have any predicted transmembrane domain.
similar membrane associated behaviour was reported when human faah was expressed as a recombinant protein lacking a n-terminal transmembrane domain and the protein was predominantly present in membrane fractions  <cit> .
discussion
bioinformatics analysis of faah amino acid sequence revealed the presence of an amidase signature domain, which is similar to that present in other mammalian faah.
the amidase signature sequence is conserved among many proteins from the amidase class, which include enzymes hydrolyzing acetamide, acrylamide, nicotinamide, and glutamide  <cit> .
faah is the only characterized mammalian enzyme belonging to the amidase class and recently the faah homolog from arabidopsis has been characterized and reported to belong to the amidase class.
despite dictyostelium faah’s considerable deviations in sequence identity across full length amino acid sequences when compared to human, porcine, rat and arabidopsis sequences, dictyostelium faah has retained anandamide hydrolysis function.
recombinant faah produced from dictyostelium and e.coli was capable of hydrolyzing anandamide and other fatty acid substrates arachidonoyl p-nitroaniline and decanoyl p-nitroaniline similar to other characterized faahs.
previously, schmid and co-workers reported n-acylethanolamine amidohydrolase from rat liver which hydrolyzed various n-acylethanolamines  <cit>  but did not test anandamide as a substrate.
later when cravatt’s group cloned and characterised n-acylethanolamine amidohydrolase cdna, the enzyme hydrolysed anandamide in addition to other fatty acid amides.
these findings indicated that the enzyme may regulate growing family of bioactive fatty acid amides, and the enzyme was renamed as fatty acid amide hydrolase.
kinetic parameters indicate that dictyostelium faah has preferred affinity for longer unsaturated acyl chains and inhibition by pmsf, ly <dig> and mafp suggest a conserved enzyme mechanism between dictyostelium and mammalian faah  <cit> .
these preliminary biochemical and kinetic analyses of dictyostelium faah supports the identification of  as a functional homolog of mammalian faah.
n-acylphosphatidylethanolamines  and its hydrolysed product n-acylethanolamines  have been previously reported in dictyostelium  <cit> .
identification of faah in dictyostelium indicates faah may be a potential regulator of naes produced in dictyostelium cells.
among many established physiological roles for anandamide in mammalian cells, recently a role in neutrophil chemotaxis was identified  <cit>  and therefore we predict a similar kind of role for naes that may exist in dictyostelium.
as recent advances are made to develop faah inhibitors for potential novel therapeutics, having a mammalian faah homolog in dictyostelium should offer an additional and moreover simple eukaryotic model system to screen any relevant drugs for their pharmacological influence at the molecular and cellular level.
CONCLUSIONS
our study indicates that dictyostelium produces anandamide hydrolysing enzyme throughout its development life cycle.
this is the first report on the identification of anandamide hydrolyzing enzyme in dictyostelium, suggesting the potential of dictyostelium as a simple eukaryotic model system to study the mechanisms of action of any faah inhibitors as drug targets.

 anandamide is a mammalian endogenous lipid that binds cannabinoid receptors which are mainly present in the central nervous system and immune cells. anandamide was identified in and named after the sanskrit word ananda, meaning bliss or delight. anandamide acts as an agonist for the central cannabinoid receptor and is therefore referred to as cannabinoid. it mimics pharmacological effects of Δ9tetrahydrocannabinol, an active ingredient of marijuana. action of anandamide is terminated by the enzyme fatty acid amide hydrolase. faah was originally identified in from rat liver plasma membrane and later faah homologs were identified from other sources including human, porcine, and arabidopsis. faah belongs to a large group of proteins containing a conserved amidase signature motif. faah can also hydrolyze, in addition to anandamide, other fatty acid derivatives like n-oleoylethanolamine and n-palmitoylethanolamine collectively referred as n-acylethanolamines. studies on mammalian faah have provided more information on naes role in regulating various physiological functions like sleep and pain. recent studies on naes reveal further biological roles in appetite suppression, vasodilatation, cardiac function and inflammation. therefore any faah inhibitors which intervene in nae’s bioactivity promise to be a novel class of therapeutics and much drug discovery research is being actively pursued in this regard. anandamide is yet to be found in dictyostelium, but its precursor n-acylphosphatidylethanolamine has previously been identified. in mammalian cells anandamide is believed to originate from hydrolysis of nape by phospholipase d. in dictyostelium, a pld homolog pldb was identified and proposed to have a similar function. identification of faah suggests that regulation of nae signalling could occur in dictyostelium and thus dictyostelium could be utilised as a simple eukaryotic model to study nae functions in parallel with mammalian systems. dictyostelium has been used to study cell motility, chemotaxis, cell differentiation and morphogenesis enabling significant contributions to an understanding of similar processes in mammalian systems. recently dictyostelium has been used as a system to study the mechanism of action for mood stabilizing drugs like lithium and valproic acid. therefore the identification of any new drug target enzyme such as faah or any drug processing mechanisms in dictyostelium suggests further potential for the use of dictyostelium in human biomedical research. dictyostelium offers an attractive system to study such processes by gene manipulation studies because of its small mbp haploid genome harbouring many homologous genes found in higher eukaryotes.  amino acid sequence analysis a putative faah in dictyostelium was identified by a bioinformatics approach searching for a human faah homolog in the dictyostelium genome. dictyostelium dna sequence ddb_g containing coding sequences for characteristic amidase signature motifs was identified and found to be located on chromosome in the annotated dictyostelium genome data base. will be referred to as dictyostelium faah as the protein’s amino acid sequence analysis and other experimental results confirm its function to be similar to mammalian faah. the calculated molecular weight of dictyostelium faah is 70 kda and domain architecture analysis reveals the presence of an amidase domain composed of a characteristic amidase signature sequence. the consensus amidase signature sequence has a conserved gssg motif shared among many proteins in the amidase class including glutamyl-t-rna amidotransferase subunit a of methanococcus jannaschii and faah from human, porcine, rat, arabidopsis and dictyostelium. faah from human, porcine and rat are composed of amino acids and faah from dictyostelium and arabidopsis contain and amino acids, respectively. faah full length protein amino acid sequence from dictyostelium lacks significant identity when compared to faah from human, porcine, rat, and arabidopsis, but identity across the amidase signature sequence increased to 40%, 38%, 38%, and 50%, for the human, procine, rat, and arabidopsis faah homologs. the serine residues at and found to be essential for rat faah activity were also conserved in as sequence of dictyostelium faah. other catalytically important residues lys ser and arg found in rat were also conserved in dictyostelium. recombinant enzyme expression and affinity purification of faah in dictyostelium and e. coli faah was expressed in dictyostelium as an n-terminal his tag fusion protein. faah was found to be predominantly a membrane associated protein and to improve yield of the purified protein, a % concentration of triton x- was used in lysis buffer to solubilise membrane fractions. cells expressing recombinant his-faah protein were solubilised in lysis buffer and subjected to ni-nta affinity chromatography separation. purified protein obtained was analyzed by coomassie staining and western blotting analysis using anti-his antibody and anti-faah polyclonal antibody respectively. initial attempts to express faah as a his tag fusion protein in e.coli were not successful, as both n-terminal his and c-terminal his fusions to faah were unstable and only a small amount of the protein was made and this was only found in inclusion bodies. alternatively, in order to simplify large scale recombinant protein production, faah was expressed and purified as a recombinant maltose binding protein fusion protein from e.coli. recombinant faah when expressed as n-terminal mbp fusion protein in e.coli produced a higher yield of soluble recombinant protein. recombinant faah when produced in either dictyostelium or e.coli migrated on sds-polyacrylamide gels, consistent with no significant post-translation modification. functional identification of dictyostelium faah dictyostelium recombinant faah expressed as n-terminal his tagged fusion protein in dictyostelium and as n-terminal mbp tagged fusion protein in e.coli hydrolyzed anandamide to free arachidonic acid and ethanolamine as determined by ce-es-ms. dictyostelium faah was also capable of hydrolyzing synthetic p-nitroanilide substrates arachidonoyl p-nitroaniline and decanoyl p-nitroaniline which were further used in kinetics studies. catalytic properties recombinant his-faah purified from dictyostelium was analyzed for fatty acid amide hydrolase activity by measuring the rate of hydrolysis of p-nitroanilide substrates apna and dpna, which were previously used to characterize binding and catalytic specificities of mammalian faah enzymes. dictyostelium faah exhibited michaelis-menten kinetics on these substrates. specific constant kcat/km values observed for apna having long chain unsaturated fatty acid were slightly higher when compared to dpna, which may indicate the enzyme’s preference for longer unsaturated acyl chains. similar observations were made with mammalian faah where the enzyme showed a fold preference for anandamide versus n-palmitoylethanolamine. the kcat values of his-faah towards apna and dpna when compared with rat faah were about and times less, respectively. purified recombinant faah enzymes from both dictyostelium and e.coli exhibited ph optima at which were similar to the mammalian faah enzymes characterized to have a ph optimum from to compounds that inhibit enzymatic activity via different mechanisms, phenylmethylsulfonyl fluoride, ly and methyl arachidonoyl fluorophosphonate were tested on dictyostelium faah in order to monitor changes in activity. non-specific irreversible serine protease inhibitor pmsf was modestly effective and inhibited about 58% at 5 mm. ly a serine hydrolase inhibitor which inactivates faah by covalent binding, was also modestly effective against dictyostelium faah and inhibited about 62% at  mm. irreversible active site targeted inhibitor mafp had potent inhibition against dictyostelium faah and inhibited about 63% at μm. identification of faah in dictyostelium the production of faah protein in dictyostelium was confirmed at the protein level. dictyostelium anti-faah polyclonal antibodies raised in rabbits were used to detect faah production during dictyostelium development. to trace the in vivo faah protein production profile, wild type dictyostelium cells allowed to develop on phosphate agar plates at different stages of development from independent single cell stage through multi-cellular fruiting body, were harvested. total proteins isolated from the harvested cells were analyzed for faah expression by western blotting using anti faah polyclonal antiserum. faah was identified as a predicted 70 kda protein expressed at constant levels throughout all the different stages of dictyostelium development suggesting an essential role for faah throughout development. however, expression levels of in vivo faah protein in dictyostelium wild type cells were very low and several attempts to study protein localization by cell fractionation and western blotting were not successful. the inability to detect endogenous faah protein in the fractionation experiments may be due to very low level of protein expression or due to protein getting degraded during the process of fractionation. therefore, ax3faah cells were used in cell fractionation studies. cells grown in liquid nutrient were harvested and fractionated into membrane and cytosol fractions by differential centrifugation. at xg, faah was distributed in both pellet and supernatant fractions indicating that faah may be a plasma membrane associated protein. at xg, faah was predominantly present in pellet fraction further indicating that faah may be associated with other intra cellular membrane bound organelles. the small quantities of faah in the supernatant after this spin strongly suggest a predominantly membrane associated protein and is further supported by increased yields of his-faah when detergents such as triton x- are added. unlike other mammalian faahs, dictyostelium faah does not have any predicted transmembrane domain. similar membrane associated behaviour was reported when human faah was expressed as a recombinant protein lacking a n-terminal transmembrane domain and the protein was predominantly present in membrane fractions. discussion bioinformatics analysis of faah amino acid sequence revealed the presence of an amidase signature domain, which is similar to that present in other mammalian faah. the amidase signature sequence is conserved among many proteins from the amidase class, which include enzymes hydrolyzing acetamide, acrylamide, nicotinamide, and glutamide. faah is the only characterized mammalian enzyme belonging to the amidase class and recently the faah homolog from arabidopsis has been characterized and reported to belong to the amidase class. despite dictyostelium faah’s considerable deviations in sequence identity across full length amino acid sequences when compared to human, porcine, rat and arabidopsis sequences, dictyostelium faah has retained anandamide hydrolysis function. recombinant faah produced from dictyostelium and e.coli was capable of hydrolyzing anandamide and other fatty acid substrates arachidonoyl p-nitroaniline and decanoyl p-nitroaniline similar to other characterized faahs. previously, schmid and co-workers reported n-acylethanolamine amidohydrolase from rat liver which hydrolyzed various n-acylethanolamines but did not test anandamide as a substrate. later when cravatt’s group cloned and characterised n-acylethanolamine amidohydrolase cdna, the enzyme hydrolysed anandamide in addition to other fatty acid amides. these findings indicated that the enzyme may regulate growing family of bioactive fatty acid amides, and the enzyme was renamed as fatty acid amide hydrolase. kinetic parameters indicate that dictyostelium faah has preferred affinity for longer unsaturated acyl chains and inhibition by pmsf, ly and mafp suggest a conserved enzyme mechanism between dictyostelium and mammalian faah. these preliminary biochemical and kinetic analyses of dictyostelium faah supports the identification of as a functional homolog of mammalian faah. n-acylphosphatidylethanolamines and its hydrolysed product n-acylethanolamines have been previously reported in dictyostelium. identification of faah in dictyostelium indicates faah may be a potential regulator of naes produced in dictyostelium cells. among many established physiological roles for anandamide in mammalian cells, recently a role in neutrophil chemotaxis was identified and therefore we predict a similar kind of role for naes that may exist in dictyostelium. as recent advances are made to develop faah inhibitors for potential novel therapeutics, having a mammalian faah homolog in dictyostelium should offer an additional and moreover simple eukaryotic model system to screen any relevant drugs for their pharmacological influence at the molecular and cellular level.  our study indicates that dictyostelium produces anandamide hydrolysing enzyme throughout its development life cycle. this is the first report on the identification of anandamide hydrolyzing enzyme in dictyostelium, suggesting the potential of dictyostelium as a simple eukaryotic model system to study the mechanisms of action of any faah inhibitors as drug targets. 
this study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from dictyostelium discoideum, suggesting the potential of dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any faah inhibitors as drug targets. wild type dictyostelium cells express faah throughout their development life cycle and the protein was found to be predominantly membrane associated. a putative faah dna sequence coding for a conserved amidase signature motif was identified in the dictyostelium genome database and the corresponding cdna was isolated and expressed as an epitope tagged fusion protein in either e.coli or dictyostelium. production of recombinant his tagged faah protein was not supported in e.coli host, but homologous dictyostelium host was able to produce the same successfully. recombinant faah protein isolated from dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates. in this study we report the identification of a faah homolog from dictyostelium discoideum and its function to hydrolyze anandamide. bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase.
 anandamide is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase. in this study we report the identification of a faah homolog from dictyostelium discoideum and its function to hydrolyze anandamide.  a putative faah dna sequence coding for a conserved amidase signature motif was identified in the dictyostelium genome database and the corresponding cdna was isolated and expressed as an epitope tagged fusion protein in either e.coli or dictyostelium. wild type dictyostelium cells express faah throughout their development life cycle and the protein was found to be predominantly membrane associated. production of recombinant his tagged faah protein was not supported in e.coli host, but homologous dictyostelium host was able to produce the same successfully. recombinant faah protein isolated from dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates.  this study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from dictyostelium discoideum, suggesting the potential of dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any faah inhibitors as drug targets. 