Molecular cloning and expression of fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.

Fatty acid alpha-hydroxylase (FAAH) catalyzes the initial reaction in alpha-oxidation of fatty acid to produce 2-hydroxy fatty acid. FAAH activity has been detected in a wide range of organisms from prokaryotes to eukaryotes. Here, we describe cloning of the FAAH gene from Sphingomonas paucimobilis, a sphingolipid- and 2-hydroxymyristic acid-rich bacterium. The isolated gene encoded 415 amino acids. A homology search revealed that amino acid sequences highly conserved in cytochrome P450 (P450) were present in FAAH. Although the heme-binding cysteine was recognizable at position 361, the consensus in the heme-binding region was modified by an insertion. Overall, FAAH has no significant identity to the known P450s. CO difference spectrum of recombinant FAAH showed the characteristic one of P450, except this peak was at 445 nm. These results suggest bacterial FAAH is a novel member of the P450 superfamily.

␣-Oxidation of fatty acids is widely observed in bacteria (1), yeasts (2), plants (3,4), and mammals (5,6). This reaction pathway results in catalysis of fatty acid to produce the corresponding fatty acid with one less carbon atom, where the fatty acid is first hydroxylated at the 2-position and sequentially decarboxylated to release carbon dioxide. This first and ratelimiting reaction is catalyzed by fatty acid ␣-hydroxylase (FAAH). 1 In mammals, the ␣-oxidation pathway is essential for catabolism of 3-methyl branched fatty acids such as phytanic acid in dietary sources like ruminant fats. Refsum disease is a human inherited disorder resulting from FAAH deficiency, in which phytanic acid exclusively accumulates in the patients' organs such as the liver and in their serum (7). In the mammalian brain, ␣-hydroxylation activity of very long chain fatty acids such as lignoceric acid has been reported to be associated with brain development, where 2-hydroxy fatty acid thus produced is introduced into cerebroside utilized for myelin formation (8,9). Using organelles isolated from liver or brain, the ␣-oxidation pathway has been investigated. However, the enzymatic properties of FAAH described in several reports were inconsistent, e.g. cofactor requirement or substrate specificity (5, 6, 10 -15). On the other hand, in plants, characterization of FAAH has been more successful. It is noted that a plant FAAH partially purified from cotyledons of germinating peanut seeds requires an H 2 O 2 -generating system consisting of glycolate oxidase for its activity (3,16,17).
We have investigated FAAH isolated from Sphingomonas paucimobilis, a bacterium containing large amounts of sphingoglycolipids (18). Most of these sphingoglycolipids contain 2-hydroxymyristic acid (19). Our previous studies indicated that FAAH from S. paucimobilis required H 2 O 2 for its activity and the oxygen atom of the H 2 O 2 was introduced into myristic acid to produce 2-hydroxymyristic acid (20). We attempted to purify the FAAH. However, complete purification of FAAH was not successful, perhaps because the amount of FAAH of S. paucimobilis was very small and its activity was too labile. Thus, we decided to clone the FAAH gene from this bacterium. Here, we describe cloning and expression of the FAAH gene from S. paucimobilis, and the characterization of the recombinant FAAH.
Isolation and Sequencing of Genomic DNA Clones Encoding FAAH-General cloning techniques were carried out essentially as described by Davis et al. (21). Genomic DNA from S. paucimobilis EY2395 T was partially digested by Sau3AI. Three-to 10-kb fragments were ligated into BamHI-digested pUC18, and these constructs were transfected into E. coli JM109. The transformed cells were cultured in Luria broth (LB) containing ampicillin. At log phase of cell growth, isopropyl-1-thio-␤-D-galactopyranoside (IPTG, Wako Pure Chemical Co., Osaka, Japan) was added into the culture medium to a final concentration of 1 mM, and the cells were cultivated for another 4 h. The cultivated cells were collected by centrifugation and disrupted by sonication in an appropriate volume of 0.1 M Tris-HCl buffer (pH 7.5). After centrifugation to remove disrupted cells, the ␣-hydroxylation activity in the resultant supernatant was assayed. For rescreening to obtain the DNA fragment encoding the N terminus of the FAAH, genomic DNA from S. paucimobilis was digested by HincII. The digested DNAs were size-selected, and 1.5-2.5-kb DNA fragments were ligated into HincII-digested pUC18, after which colony hybridization was performed. Labeling of the probe and detection of hybridized fragments were performed with the digoxigenin DNA labeling and detection kit (Boehringer Mannheim, Mannheim, Germany).
Sequencing of DNA fragments was performed using the dye-terminator cycle sequencing kit and an ABI 373A DNA sequencer (Perkin-Elmer, Foster City, CA). Alignment analysis was performed as described by Gotoh (22).
Overexpression, Purification, and Characterization of FAAH Protein-An EcoRI blunt-ended XbaI fragment containing the FAAH gene was ligated into EcoRI, SmaI-digested pGEM-4T-3. The construct was * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
transfected into E. coli BL21, and transformed cells were cultured in LB containing ampicillin at 25°C. At log phase, IPTG was added to the culture medium to a final concentration of 0.1 mM. After another 20 h of cultivation, cells were collected by centrifugation and disrupted by sonication in 0.1 M Tris-HCl buffer (pH, 7.5), 20% ethylene glycol, 1 mM dithiothreitol (buffer A) containing 1% cholic acid, 0.1% SDS, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride (Nacalai Tesque, Inc., Kyoto, Japan). The supernatant obtained by centrifugation at 100,000 ϫ g for 60 min was diluted by addition of 2 volumes of buffer A. Glutathione S-transferase (GST)-FAAH fusion protein was bound to glutathione-Sepharose (Pharmacia) and then eluted with buffer A containing 0.3% cholic acid, 0.03% SDS, 1.5 M urea, and 5 mM reduced glutathione. The eluate was treated with thrombin at 25°C for 20 h. The resultant eluate was dialyzed against 0.1 M sodium phosphate buffer (pH 7.0), 30% ethylene glycol, 0.4% cholic acid, and 1 mM dithiothreitol (buffer B). The dialyzed eluate was applied to a hydroxylapatite KB HPLC column (Koken Co., Ltd., Tokyo, Japan) equilibrated with buffer B. After the column was washed with an excess amount of buffer B, FAAH was eluted with a 0.1-0.45 M sodium phosphate gradient. CO difference spectrum of the purified FAAH was determined by the method of Omura and Sato (23).
Determination of ␣-Hydroxylation Activity and Identification of Product-The assay for fatty acid ␣-hydroxylation was performed as described previously (20), except sodium azide was added into the reaction mixture to a final concentration of 3 mM. For identification of the product, the reaction mixture contained 0.1 M Tris-HCl buffer (pH 8.0), 0.2 mM H 2 O 2 , 60 M myristic acid, and 0.5 g of purified FAAH in a total volume of 0.2 ml. Incubation was performed at 37°C for 10 min. product were extracted with ethyl acetate and treated with 9-anthryldiazomethane (Funakoshi Chemical Co., Tokyo, Japan). The 9-anthryldiazomethane-derivatized fatty acids were analyzed by HPLC as described previously (24).

RESULTS AND DISCUSSION
For cloning of the FAAH gene, a genomic library was constructed using pUC18 and E. coli JM109 transfected with the constructed plasmids was cultivated. After induction by IPTG, the cells were collected, disrupted by sonication, and centrifugated, and then the ␣-hydroxylation activity in the resultant supernatant was assayed. Four thousand clones were screened, and one positive clone was obtained. The plasmid designated pOC4 isolated from this clone contained a 3.3-kb insert (OC4) (Fig. 1). Sequence analysis of this insert revealed two significant open reading frames (ORFs) at 5Ј-and 3Ј-ends. We found that the coding sequence of lacZ of the vector plasmid was connected in frame to 5Ј-ORF of OC4. Deletion mutants at the 5Ј-end or 2.8 kb from the 3Ј-end abolished ␣-hydroxylation activities. Therefore, we concluded that the 5Ј-ORF encoded the FAAH. Surprisingly, a homology search for the deduced amino acid sequence of the 5Ј-ORF revealed homology to sequences conserved in cytochrome P450 (P450); in particular, a highly homologous sequence to the aromatic region of P450 (25) was found in FAAH (Fig. 2). Alignment analysis for FAAH and the known P450s suggested that the N-terminal of FAAH was missing, and thus we screened the library constructed with HincII-digested genomic DNA using a 513-bp fragment from the 5Ј-end of OC4 (from Sau3AI at 5Ј-end of OC4 to HincII (SalI)) as a probe (Fig. 1). We obtained a 2.4-kb fragment and found the initiation codon at 54 bp upstream from the 5Ј-end of OC4. A termination codon was located in frame 51 bp upstream from the initiation codon ( Fig. 2A). The sequence from Ϫ29 to Ϫ24, AAGGAG, matched to the Shine-Dalgarno consensus sequence, although it was so far from the initiation codon. Consequently, the FAAH gene encoded a protein of 415 amino acids and the calculated molecular weight was 46,485.
As mentioned above, significant homology to the highly conserved regions among the P450 superfamily was observed. Particularly, the sequence of the aromatic region of FAAH was highly homologous to that of P450: 75% identical to that of CYP 3A5 (26) (Fig. 2C). With the exception of two cases, Bacillus megaterium CYP 102A and Anabaena sp. ORF3-encoded sequence, bacterial P450s that belong to the B-class generally lack the aromatic region, while all mammalian P450s that belong to the E-class have the aromatic region (25). Therefore, the bacterial FAAH reported here constitutes a third case belonging to the E-class. In helix-K, the EXXR motif, which is completely conserved in the P450 superfamily, was found in FAAH (Fig. 2B). Although the heme-binding cysteine was found at position 361, the consensus motif of the heme-binding region, FXXGXXXCXG, was modified, where conserved phenylalanine was substituted to glutamine and seven amino acids were found between conserved glycine and cysteine residues (Fig. 2D). Such insertions were also found in plant P450s, CYP 74A1 (27) and CYP 74B1 (28), in which the phenylalanine was substituted by tryptophan. Interestingly, these P450 enzymes utilized peroxy substrates and did not require molecular oxygen and reducing equivalents for their activities. We observed that the bacterial FAAH efficiently utilized lower amounts of H 2 O 2 , and in the presence of H 2 O 2 , molecular oxygen and reducing equivalents were not required for ␣-hydroxylation (20), suggesting that the structural similarity among FAAH and these peroxide-metabolizing P450s may affect their unusual enzymatic properties. Moreover, in addition to these peroxide-metabolizing P450s, the other P450s, CYP 5A1 (29) and CYP 8A1 (30), which metabolize an endoperoxide, prostag-landin H 2 , and also do not require molecular oxygen, have substitution of threonine at the appropriate position in helix-I, which is believed to be the critical residue for O 2 activation (31). The sequence of helix-I of FAAH showed no significant homology to those of other P450s and also lacked an adjacent threonine residue. These findings also supported the hypothesis that FAAH utilizes H 2 O 2 but dose not require O 2 activation by reductase such as NADPH-cytochrome P450 reductase (20). Despite the high degree of conservation of sequences found in P450s, overall FAAH had no significant identity to the known P450s (less than 25% identity). However, the hydrophobicity profile (32) of FAAH was similar to those of other bacterial P450s, P450 cam (CYP 101A) and P450 BM3 (CYP 102A) hydroxylase domain, which are soluble enzymes (33) (Fig. 3), supporting the observation that FAAH was isolated in the soluble fraction (18).
To examine whether the FAAH gene-encoded protein has the properties of P450, we purified the FAAH overexpressed in E. coli. As shown in Fig. 4A, the EcoRI-XbaI fragment of pOC4 was inserted into the pGEM-4T-3 expression vector to construct a GST-FAAH fusion gene. In this construct, FAAH was truncated by 18 amino acids at N terminus, but contained all the elements necessary for its activity. The expressed fusion protein was isolated with glutathione-Sepharose and digested with thrombin, and then the FAAH was purified by HPLC with a hydroxylapatite column (Fig. 4B). The purified FAAH showed myristic acid ␣-hydroxylation activity in the presence of H 2 O 2 to produce 2-hydroxymyristic acid, while no such activity was detected in the absence of H 2 O 2 (Fig. 4C). The K m value for H 2 O 2 was approximately 60 M, which was similar to that of native FAAH partially purified from S. paucimobilis. Other peroxy compounds, cumene hydroperoxide, t-butyl hydroperoxide, and t-butyl peroxybenzonate, were not effective (data not shown). We observed that, like the recombinant FAAH, native FAAH was specific for H 2 O 2 . The turnover rate of myristic acid ␣-hydroxylation by FAAH was very high, comparable to that of P450 BM3 , which efficiently catalyzes lauric acid (-2)-hydroxylation (34); specific activity of FAAH was 838 nmol/min/nmol of protein. We next examined the spectral properties of the FAAH. The CO difference spectrum of FAAH showed the char-acteristic one of P450, except this peak was at 445 nm (Fig. 4D). On the basis of the results of sequence and spectral analyses of FAAH, we concluded that bacterial FAAH was a novel member of the cytochrome P450 superfamily.
Previously, Muralidharan and Kishimoto (14) showed partial inhibition of phytanic acid ␣-oxidation of rat liver by CO, indicating a possible involvement of P450. Recently, Pahan et al. (35) reported that CO inhibited phytanic acid ␣-hydroxylation activity of peroxisomes from human liver. Despite the analysis of CO difference spectrum of the bacterial FAAH revealed that  5 g of protein). C, identification of the product (2-hydroxymyristic acid) by HPLC. The peaks at retention times of 9.5, 13, and 23 min corresponded with those of cholic acid, 2-hydroxymyristic acid, and myristic acid, respectively. The asterisk shows the peak of 2-hydroxymyristic acid. Incubation was carried out with H 2 O 2 for 0 min (a), without H 2 O 2 for 10 min (b), and with H 2 O 2 for 10 min (c). D, CO difference spectrum of purified FAAH. The specific content of P450 was 9.8 nmol/mg of protein. Note that the peak was at 445 nm. Fe(II)-CO complex was formed (Fig. 4D), the inhibition of the hydroxylation activity by CO, as can be seen in most of P450s, was not observed (data not shown).
Most recently, Borge et al. (36) reported that palmitic acid can be ␣-oxidized to release CO 2 by only one enzyme purified from cucumber, although its purification was not complete, judging from the observation on SDS-polyacrylamide gel electrophoresis. If fatty acids were first ␣-hydroxylated, and then decarboxylated in ␣-oxidation, this enzyme must catalyze these two reactions. FAAH described in this report, however, did not show significant decarboxylase activity. Therefore, at least in bacteria, decarboxylase and FAAH could exist separately. Furthermore, there seems to be a resemblance between the bacterial FAAH and a plant FAAH (16,17). Both enzymes catalyze ␣-hydroxylation of straight chain fatty acids and require an H 2 O 2 -generating system for their activities. It is particularly interesting that the plant peroxide-metabolizing P450s (27,28) show the structural similarity to the bacterial FAAH in the heme-binding region, suggesting that P450-like enzymes may involve in fatty acid ␣-hydroxylation, at least in some species of plants. In any case, further studies are necessary to clarify these points.