Other NAcylethanolamine Amidases

As noted, recent experimental evidence has clearly demonstrated that Arabidopsis FAAH encoded by the gene At5g64440 is a bonafide NAE hydrolase (Shrestha et al. 2003, 2006; Wang et al. 2006). The ability of Arabidopsis FAAH knockouts to deplete their endogenous seed NAEs during germination, however, point to the existence of alternative pathways for NAE catabolism in plants (Wang et al. 2006). Although it has yet to be determined whether plants have other enzymes with NAE hydrolytic activity, a FAAH-2 enzyme was recently identified in human cancer cell lines via activity-based protein profiling. The human FAAH-2 protein was shown to share 20% sequence similarity with FAAH-1 and comparison of the enzymatic properties of FAAH-1 and FAAH-2 revealed differences in substrate selectivity. For example, FAAH-1 was more active in hydrolyzing NAE18:1, NAE16:0, and anandamide (NAE20:4) compared to FAAH-2. FAAH-2 also was unable to hydro-lyze other fatty acid amides such as the N-acyl taurines (NAT), which are substrates of FAAH-1. Thus, it appears that differences in substrate selectivity between the two animal FAAHs are dictated by the amine leaving group and degree of acyl chain saturation (Wei et al. 2006). The precise mechanisms by which FAAH-1 and FAAH-2 coordinate their activities to regulate NAE metabolism and signaling remain to be elucidated.

Another enzyme capable of hydrolyzing NAEs was recently identified in animals. This enzyme designated as NAE acid amidase (NAAA) had no homology to FAAH-1 and was more closely related to the family of acid ceramidase proteins. Not surprisingly, the catalytic properties of NAAA differed significantly from that of FAAH-1 in that NAAA was most active at a pH range of 4.5-5 while FAAH-1 was more active at a broader pH range with optimal activity at more alkaline pH (Tsuboi et al. 2005). Substrate preferences of NAAA and FAAH-1 were also different in that FAAH-1 was highly reactive to anandamide (NAE20:4), NAE18:2, and NAE16:0 while NAAA hydrolyzed NAE14:0 with greater efficiency (Tsuboi et al. 2005). Furthermore, consistent with its preference for acidic environments, NAAA was shown to be localized at lysosomes in animal cells (Tsuboi et al. 2005, 2007). NAAA is widely distributed in various organs of mice and could degrade various NAEs in macrophages in cooperation with FAAH-1 (Sun et al. 2005). Like the recently discovered FAAH-2, additional studies will be needed to determine the precise physiological role of NAAA in NAE metabolism and how it contributes toward defining overall NAE profiles in the cell.

No close homology of the animal NAAA and FAAH-2 enzymes could be identified in plant databases. Other plant genes, however, encode for proteins with amidase signature domains that could be a possible NAE hydrolases. Among the seven amidase signature proteins in the Arabidopsis genome, only AtFAAH and amidase 1 (AT-AMI1) have been characterized (Kilaru et al. 2007). Unlike AtFAAH, which efficiently hydrolyzed NAEs, AT-AMI1 was highly specific for indole-3-acetamide and 1-naphthaleneacetamide, and exhibited only minimal hydrolytic activity toward NAEs (Pollmann et al. 2006). Furthermore, while

AtFAAH appears to be a bifunctional enzyme by having both amidase and esterase activity (Shrestha et al. 2006), AMI-1 had no detectable esterase activity (Neu et al. 2007). The in vitro enzymatic properties of the other five plant amidases have yet to be determined but expression data from microarray studies are pointing toward potential coordination of some of these amidases with AtFAAH. The At5g07360 gene, for example, is a particularly intriguing candidate amidase because its in silico expression patterns, as determined from gene chip data from Genevestigator, show high expression in dessicated seeds (Zimmermann et al. 2004). Furthermore, this gene is one of the most highly expressed in seeds of the ABA hypersensitive mutant ahg1 (Nishimura et al. 2007). Since recent data shows that NAE interacts with ABA signaling during seed germination and early seedling development (Teaster et al. 2007), detailed studies of the amidase encoded by the At5g07360 should provide additional insights into the impact of NAE catabolism on plant stress responses. Similar to AtFAAH, knockouts to the At5g07360 gene display hypersensitivity to the growth inhibitory effects of exogenous NAE12:0, indicating that this gene could contribute to NAE degradation in planta (Blancaflor and Chapman, unpublished observations). Expression of the amidase encoded by the At5g07360 gene in a heterologous system combined with genetic analysis of plants altered in expression of this potential amidase provide exciting avenues for future research.

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