Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide. FAAH's primary structure identifies this enzyme as a member of a diverse group of alkyl amidases, known collectively as the "amidase signature family". At present, this enzyme family's catalytic mechanism remains poorly understood. In this study, we investigated the catalytic features of FAAH through mutagenesis, affinity labeling, and steady-state kinetic methods. In particular, we focused on the respective roles of three serine residues that are conserved in all amidase signature enzymes (S217, S218, and S241 in FAAH). Mutation of each of these serines to alanine resulted in a FAAH enzyme bearing significant catalytic defects, with the S217A and S218A mutants showing 2300- and 95-fold reductions in k(cat), respectively, and the S241A mutant exhibiting no detectable catalytic activity. The double S217A:S218A FAAH mutant displayed a 230 000-fold decrease in k(cat), supporting independent catalytic functions for these serine residues. Affinity labeling of FAAH with a specific nucleophile reactive inhibitor, ethoxy oleoyl fluorophosphonate, identified S241 as the enzyme's catalytic nucleophile. The pH dependence of FAAH's k(cat) and k(cat)/K(m) implicated a base involved in catalysis with a pK(a) of 7.9. Interestingly, mutation of each of FAAH's conserved histidines (H184, H358, and H449) generated active enzymes, indicating that FAAH does not contain a Ser-His-Asp catalytic triad commonly found in other mammalian serine hydrolytic enzymes. The unusual properties of FAAH identified here suggest that this enzyme, and possibly the amidase signature family as a whole, may hydrolyze amides by a novel catalytic mechanism.

Early work on aminoacylation of alanine-specific tRNA (tRNA(Ala)) by alanyl-tRNA synthetase (AlaRS) gave rise to the concept of an early "second genetic code" imbedded in the acceptor stems of tRNAs. A single conserved and position-specific G:U base pair in the tRNA acceptor stem is the key identity determinant. Further understanding has been limited due to lack of a crystal structure of the enzyme. We determined a 2.14 A crystal structure of the 453 amino acid catalytic fragment of Aquifex aeolicus AlaRS. It contains the catalytic domain characteristic of class II synthetases, a helical domain with a hairpin motif critical for acceptor-stem recognition, and a C-terminal domain of a mixed alpha/beta fold. Docking of tRNA(Ala) on AlaRS shows critical contacts with the three domains, consistent with previous mutagenesis and functional data. It also suggests conformational flexibility within the C domain, which might allow for the positional variation of the key G:U base pair seen in some tRNA(Ala)s.

The genetic code is defined by the specific aminoacylations of tRNAs by aminoacyl‐tRNA synthetases. Although the synthetases are widely conserved through evolution, aminoacylation of a given tRNA is often system specific—a synthetase from one source will not acylate its cognate tRNA from another. This system specificity is due commonly to variations in the sequence of a critical tRNA identity element. In bacteria and the cytoplasm of eukaryotes, an acceptor stem G3:U70 base pair marks a tRNA for aminoacylation with alanine. In contrast, Drosophila melanogaster (Dm) mitochondrial (mt) tRNAAla has a G2:U71 but not a G3:U70 pair. Here we show that this translocated G:U and the adjacent G3:C70 are major determinants for recognition by Dm mt alanyl‐tRNA synthetase (AlaRS). Additionally, G:U at the 3:70 position serves as an anti‐determinant for Dm mt AlaRS. Consequently, the mitochondrial enzyme cannot charge cytoplasmic tRNAAla. All insect mitochondrial AlaRSs appear to have split apart recognition of mitochondrial from cytoplasmic tRNAAla by translocation of G:U. This split may be essential for preventing introduction of ambiguous states into the genetic code.

Plants and certain protists use cycloeucalenol cycloisomerase (EC 5.5.1.9) to convert pentacyclic cyclopropyl sterols to conventional tetracyclic sterols. We used a novel complementation strategy to clone a cycloeucalenol cycloisomerase cDNA. Expressing an Arabidopsis thaliana cycloartenol synthase cDNA in a yeast lanosterol synthase mutant provided a sterol auxotroph that could be genetically complemented with the isomerase. We transformed this yeast strain with an Arabidopsis yeast expression library and selected sterol prototrophs to obtain a strain that accumulated biosynthetic ergosterol. The novel phenotype was conferred by an Arabidopsis cDNA that potentially encodes a 36-kDa protein.We expressed this cDNA (CPI1) in Escherichia coli and showed by gas chromatography-mass spectrometry that extracts from this strain isomerized cycloeucalenol to obtusifoliol in vitro. The cDNA will be useful for obtaining heterologously expressed protein for catalytic studies and elucidating the in vivo roles of cyclopropyl sterols.Even distantly diverged organisms generally biosynthesize shared molecules by such similar routes that bacteria and yeast have been suitable systems for deducing much of human metabolism. Sterol biosynthesis provides an exception to this trend. Eukaryotes use two distinct pathways ( Fig. 1) to make structurally similar sterols (1). Animals and fungi cyclize oxidosqualene to the tetracyclic ⌬ 8 -triterpene lanosterol (2), which they metabolize further to tetracyclic membrane sterols (3)(4)(5). Plants cyclize instead to the pentacyclic cycloartenol (6, 7) and consequently must isomerize the cyclopropane ring to form ⌬ 8 tetracycles en route to tetracyclic sterols. Why plants use this two-enzyme route (8) rather than cyclizing directly to tetracyclic sterols using lanosterol synthase remains unknown.Although the enzyme has not been purified to homogeneity, cycloeucalenol cycloisomerase (also known as cycloeucalenolobtusifoliol isomerase) activity has been observed directly in both dicots (9) and monocots (10). In addition, the presence of an isomerase has been demonstrated indirectly in diverse plants (11)(12)(13)(14) and the protozoan Acanthamoeba polyphaga (15) by identifying cyclopropyl sterols after treatment with isomerase inhibitors. In D 2 O, the isomerase adds a deuterium to C-19 and abstracts hydrogen from C-8 (16, 17). Neither ATP nor NADPH enhances activity (8), and acidic ring opening seems the most likely mechanism. Because the nonenzymatic acid-catalyzed isomerization (18) requires severe conditions (10% H 2 SO 4 in refluxing isopropyl alcohol for 24 h), it is likely that the enzyme employs a metal or an unusually acidic amino acid residue to open the ring.As a prelude to investigating its catalytic mechanism and biological function, we have cloned and heterologously expressed the Arabidopsis thaliana cDNA that encodes cycloeucalenol cycloisomerase. EXPERIMENTAL PROCEDURESCloning the Cycloeucalenol Cycloisomerase cDNA-The A. thaliana CAS1 1 cDNA (19) cloned into the integrative yeast exp...

Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50-52% identical to known higher plant cycloartenol synthases and 40-49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.