Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae) Koji Noge 1, *, Makiko Kato 1 , Naoki Mori 1 , Michihiko Kataoka 1 , Chihiro Tanaka 2 , Yuji Yamasue 3 , Ritsuo Nishida 1 and Yasumasa Kuwahara 1, † 1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan 2 Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Japan 3 Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Japan Keywords alarm pheromone; biosynthesis; Carpoglyphus lactis; geraniol dehydrogenase; monomeric alcohol dehydrogenase Correspondence K. Noge, Department of Entomology, University of Arizona, Tucson, AZ 85721, USA Fax: +1 520 621 1150 Tel: +1 520 621 1328 E-mail: noge@email.arizona.edu Present address *Department of Entomology, University of Arizona, Tucson, AZ, USA †Department of Bioscience and Biotech- nology, Faculty of Bioenvironmental Science, Kyotogakuen University, Kameoka, Japan Database Nucleotide sequence data are available in the DDBJ ⁄ EMBL ⁄ GenBank databases under the accession numbers AB305641 and AB305642 (Received 30 October 2007, revised 20 March 2008, accepted 25 March 2008) doi:10.1111/j.1742-4658.2008.06421.x Geraniol dehydrogenase (GeDH), which plays an important role in the bio- synthesis of neral, an alarm pheromone, was purified from the astigmatid mite Carpoglyphus lactis. The enzyme was obtained in an apparently homo- geneous and active form after 1879-fold purification through seven steps of chromatography. Car. lactis GeDH was determined to be a monomer in its active form with a relative molecular mass of 42 800, which is a unique subunit structure in comparison with already established alcohol dehydro- genases. Car. lactis GeDH oxidized geraniol into geranial in the presence of NAD + . NADP + was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD + -dependent alcohol dehydrogenase. The optimal pH and temperature for geraniol oxidation were determined to be pH 9.0 and 25 °C, respectively. The K m values for geraniol and NAD + were 51.0 lm and 59.5 lm, respectively. Car. lactis GeDH was shown to selec- tively oxidize geraniol, whereas its geometrical isomer, nerol, was inert as a substrate. The high specificity for geraniol suggests that Car. lactis GeDH specializes in the alarm pheromone biosynthesis of Car. lactis. Car. lactis GeDH is composed of 378 amino acids. Structurally, Car. lactis GeDH showed homology with zinc-dependent alcohol dehydrogenases found in mammals and a mosquito (36.6–37.6% identical), and the enzyme was considered to be a member of the medium-chain dehydrogenase ⁄ reductase family, in view of the highly conserved sequences of zinc-binding and NAD + -binding sites. Phylogenetic analyses indicate that Car. lactis GeDH could be categorized as a new class, different from other established alcohol dehydrogenases. Abbreviations ADH, alcohol dehydrogenase; CAD, cinnamyl alcohol dehydrogenase; GeDH, geraniol dehydrogenase; MDR, medium-chain dehydrogenase ⁄ reductase. FEBS Journal 275 (2008) 2807–2817 ª 2008 The Authors Journal compilation ª 2008 FEBS 2807 Communication in insects and other arthropods, including mites, is mostly established by chemical sig- nals. Pheromones play an essential role in communica- tive signals between individuals of the same species in relation to mating, aggregation, escape, and other behaviors. Since bombykol [( E,Z)-10,12-hexadecadien- 1-ol] was isolated as a sex pheromone of the silkmoth, Bombyx mori [1], a wide variety of chemicals, such as terpenoids, fatty acid derivatives (alcohols, aldehydes, esters, and hydrocarbons), phenolics, and alkaloids, have been identified as pheromones from thousands of insects, mites, and other arthropods. Astigmatid mites secrete various kinds of monoterp- enes, such as neral [(Z)-3,7-dimethyl-2,6-octadienal], (2R,3R)-epoxyneral [(2R,3R)-epoxy-3,7-dimethyl-6- octenal] and a-acaridial [2(E)-(4-methyl-3-pentenyl)- butenedial], and some of these compounds are known to function as alarm, aggregation and sex pheromones [2]. Neral has the basic skeleton of most of the mite monoterpenes. Because of this, neral is thought to be a key compound in the monoterpene biosynthesis of mites. Neral is one of the most widely distributed mite monoterpenes. It is present in 35 of 60 species of astig- matid mites examined, including 28 unidentified species [2]. Neral has been identified as the alarm pheromone in six mite species, including Carpoglyphus lactis [3–7]. This compound was also identified as the aggregation pheromone of Schwiebea elongata [8] and as the sex pheromone of Histiogaster sp. [9]. Furthermore, it was reported that a mixture of neral and geranial [(E)-3,7- dimethyl-2,6-octadienal] secreted from astigmatid mites had an antifungal effect on Aspergillus fumigatus [10]. From these facts, it has been suggested that neral plays important roles in the survival of astigmatid mites in relation to chemical communication and defense. In Car. lactis, neral is biosynthesized via the mevalonate pathway [11]. A recent study showed that neral was biosynthesized from geraniol in Car. lactis [12]: gera- niol was first oxidized to geranial by NAD + -depen- dent geraniol dehydrogenase (GeDH; EC 1.1.1.183), and then geranial was isomerized to neral (Fig. 1). In general, neral and geranial are present as a mixture in thermodynamic equilibrium (citral; neral ⁄ geranial = 4 : 6). In the secretion of Car. lactis, however, the neral ⁄ geranial ratio is 95 : 5 and is thus different from that of citral [3,12]. Although in this previous study large amounts of neral were not produced in vitro, the enzyme solution obtained from Car. lactis converted citral into a different composition of isomers (geranial ⁄ neral = 1 : 1). Therefore, it suggested that there was some enzymatic factor related to the isomerization from geranial to neral in Car. lactis [12]. The activities of GeDH have been reported in four plant materials (lemongrass, Cymbopogon flexuosus [13], orange fruit [14], ginger, Zingiber officinale [15], and sweet basil, Ocimum basilicum [16]), and two microorganisms (Pseudomonas aeruginosa [17] and Penicillium digitatum [18]). However, there is only one example of the pres- ence of GeDH in an animal source, i.e. Car. lactis [12]. Therefore, the enzymatic characteristics of animal GeDH and its relationship to other GeDHs are still unknown. This article reports the first purification to homoge- neity of GeDH from an animal. We elucidated its enzymatic characteristics associated with the alarm pheromone biosynthesis. We also report the nucleotide and amino acid sequences of Car. lactis GeDH. On the basis of the comparison of enzymatic properties and protein sequences, Car. lactis GeDH is suggested to be a new class, differing from the already estab- lished alcohol dehydrogenases (ADHs). This is also the first report of the primary structure determination of GeDH from an animal. Results Purification of GeDH from Car. lactis Car. lactis GeDH was purified to homogeneity via a seven-step protocol, which is summarized in Table 1. The final recovery of Car. lactis GeDH was 1.4% with 1879-fold purification. Gel filtration chromatography on a Superdex 75 column of the purified enzyme resulted in a single peak of protein, corresponding to GeDH activity, with a calculated relative molecular Fig. 1. Biosynthetic pathway of neral in Car. lactis. Geraniol, which is biosynthesized via the mevalonate pathway [11], is first oxidized to geranial by NAD + -dependent geraniol dehydrogenase (1), and then geranial is isomerized to neral [12]. Nerol is not oxidized directly to neral [12]. Geraniol dehydrogenase from Carpoglyphus lactis K. Noge et al. 2808 FEBS Journal 275 (2008) 2807–2817 ª 2008 The Authors Journal compilation ª 2008 FEBS mass of 56 200. Native PAGE of the purified enzyme gave a single band (data not shown). SDS ⁄ PAGE of the purified enzyme showed a single band, with a cal- culated relative molecular mass of 42 800 (Fig. 2). No other bands corresponding to the difference (about 13 000) between native and denatured enzymes were detected by SDS ⁄ PAGE. In addition to this, the rela- tive molecular mass of the native enzyme was less than that expected for a homodimer. These observations suggest that Car. lactis GeDH may be active as a monomer. Catalytic properties for geraniol oxidation Purified Car. lactis GeDH required only NAD + for the oxidation of geraniol. NADP + was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD + -dependent ADH. The effect of temperature on the geraniol oxidation activity is shown in Fig. 3. The optimal temperature was estimated to be 25 °C. The geraniol oxidation activity increased under basic condi- tions (Fig. 4). As the pH increased from 7.0 to 7.5, there was a sharp increase in GeDH activity, whereas Table 1. Purification of GeDH from Car. lactis. Purification step Total protein (mg) Total activity (units) Specific activity (unitsÆmg )1 ) Yield (%) Purification (fold) Crude enzyme 7233 119 0.016 100 1.0 (NH 4 ) 2 SO 4 precipitate 4913 87 0.018 73 1.1 TOYOPEARL Phenyl-650M 100 24 0.24 20 15 TOYOPEARL HW-50F 70 11 0.16 9.2 9.6 DEAE–cellulose 8.9 9.4 1.1 7.9 64 Blue-Cellulofine 0.88 6.4 7.3 5.4 442 Superdex 200 0.35 3.6 10 3.0 625 MonoQ 0.055 1.7 31 1.4 1879 Fig. 2. SDS ⁄ PAGE of purified Car. lactis GeDH. Lane 1, standard mixture (see Experimental procedures); lane 2, purified enzyme. Proteins were stained with silver reagent. Fig. 4. pH activity profile of purified Car. lactis GeDH. Activity was measured using 0.1 m M NAD + and 0.1 mM geraniol. The buffers used were: 50 m M potassium dihydrogen citrate ⁄ NaOH (pH 4.0–6.0); 50 m M potassium dihydrogen phosphate ⁄ NaOH (pH 6.0–7.5); 50 m M Tris ⁄ HCl (pH 7.0–8.5); and 50 mM glycine ⁄ NaOH (pH 8.5–10.5). The geraniol oxidation activity was inhibited in potassium phosphate buffer. Fig. 3. Temperature dependence of geraniol oxidation by purified Car. lactis GeDH. The oxidation activities at different temperatures were estimated as consumption of the substrate, geraniol. K. Noge et al. Geraniol dehydrogenase from Carpoglyphus lactis FEBS Journal 275 (2008) 2807–2817 ª 2008 The Authors Journal compilation ª 2008 FEBS 2809 the activity decreased sharply beyond pH 10.0. The optimal pH was determined to be 9.0. The enzyme exhibited typical Michaelis–Menten kinetics with gera- niol as well as NAD + . The K m values for geraniol and NAD + were determined from a Lineweaver–Burk plot (data not shown) as 51.0 lm and 59.5 lm, respectively. The affinity of the enzyme for geraniol was almost the same as that for NAD + . k cat and k cat ⁄ K m values for geraniol were 2996 s )1 and 58.8 s )1 Ælm )1 . To calculate the k cat value, the relative molecular mass of the native enzyme was calculated from the deduced amino acid sequence (40 630, described below). Substrate specificity The substrate specificity of purified Car. lactis GeDH is shown in Table 2. Geraniol, a trans-allylic alcohol, was selectively oxidized by the enzyme. Nerol, the geo- metrical isomer of geraniol, was inert as a substrate. Saturation of the 2,3-double bond of geraniol, giving citronellol, resulted in a marked decrease in the oxida- tion activity. These results indicate that Car. lactis GeDH could recognize the geometrical isomers clearly. An increase of one in the number of isoprene units of geraniol, giving E,E-farnesol, resulted in a drastic decrease in the oxidation activity (50%). The relative activity was 4.3% for 3-methyl-2-buten-1-ol, which corresponds to an allylic alcohol with one isoprene unit. Straight-chain alcohols such as 1-octanol, 2-buten-1-ol, 1-butanol and ethanol served as poor substrates. These results suggest that the enzyme can recognize allylic alcohols with carbon chain lengths of at least more than five. Sequence analysis A sequence of 42 N-terminal amino acid residues of purified Car. lactis GeDH was identified as shown in Fig. 5. The N-terminus (valine) was detected by Edman degradation. It is suggested that at least valine as the first residue was not blocked by acetylation. The N-terminal residues of the enzyme shared 38.1% simi- larity to two plant ADHs: tomato, Solanum lycopersi- cum (Swiss-Prot ⁄ TrEMBL primary accession number Q41242), and potato, Solanum tuberosum (Q43169), by blast search. A specific degenerate primer was designed on the basis of the N-terminal amino acid sequence at position 18–25 (see Experimental proce- dures). The degenerate primer used in PCR generated a cDNA fragment encoding the enzyme with a size of approximately 1.1 kbp (Fig. 5). On the basis of the cDNA sequence, gene-specific primers were designed, and then the nucleotide sequence corresponding to the N-terminal residues of purified Car. lactis GeDH at position 1–32 was determined by inverse PCR using genomic DNA (Fig. 5). The N-terminal sequence deduced from this nucleotide sequence completely matched with that determined by Edman degradation, and no intron was detected in this region. Although the residues at positions 11, 35 and 40 were not identi- fied by Edman degradation, these residues were identi- fied as a cysteine, a proline, and a valine, respectively, on the basis of the deduced amino acid sequence. Car. lactis GeDH was composed of 378 amino acids and its relative molecular mass was 40 630, which approximately corresponds to the 42 800 determined by SDS ⁄ PAGE. The blast search revealed that the deduced amino acid sequence of Car. lactis GeDH has similarity to the zinc-dependent ADHs found in vari- ous kinds of vertebrates (36.7% identical to ostrich, Struthio camelus, class II, P80468; 36.6% identical to Norway rat, Rattus norvegicus, class III, P12711; 37.6% identical to Japanese medaka, Oryzias latipes, class III, Q6R5I9), and a yellow fever mosquito, Aedes aegypti (37.6% identical, Q176A7). The ATG sequence was found next to the first valine of the puri- fied enzyme, which may correspond to an initiator methionine. A typical TATA box was not detected in the 201 bp upstream from the probable initiator codon (data not shown; see AB305642 deposited in the DDBJ ⁄ EMBL ⁄ GenBank databases). Phylogenetic analysis The protein distance tree was constructed by using protein sequences related in both primary structure and substrate specificity to Car. lactis GeDH: ADHs, cinnamyl alcohol dehydrogenases (CADs), Oc. basili- cum GeDH, and two 10-hydroxygeraniol oxidoreduc- tases, as shown in Fig. 6. ADHs found in microorganisms (yeast ADHs and Neurospora ADH) Table 2. Substrate specificity of Car. lactis GeDH. Activities were determined in 50 m M Tris ⁄ HCl (pH 8.5), in the presence of 0.1 mM NAD + and 0.1 mM each alcohol. Substrate Relative activity (%) Geraniol 100 Nerol 0.0 Citronellol 4.3 E,E-Farnesol 50 3-Methyl-2-buten-1-ol 4.3 1-Octanol 3.7 1-Butanol 2.5 2-Buten-1-ol 3.1 Ethanol 1.2 Geraniol dehydrogenase from Carpoglyphus lactis K. Noge et al. 2810 FEBS Journal 275 (2008) 2807–2817 ª 2008 The Authors Journal compilation ª 2008 FEBS were used as outgroups. The tree was separated into three major groups, and the monophyly of each was supported by a high bootstrap value. The first group consisted of ADHs obtained from both animals and plants. The second group consisted of two groups: plant CADs and three other geraniol-related dehydro- genases. The third group consisted of Car. lactis GeDH. The topologies of the trees obtained from both maximum parsimony and minimum evolution criteria were congruent, except for the position of Oryzias Fig. 5. Nucleotide and deduced amino acid sequences of Car. lactis GeDH. The single- letter code translated amino acid sequence is indicated below the nucleotide sequence from positions 1 to 1134 (as 378 amino acids). Nucleotide and amino acid positions are indicated on the right and left, respec- tively. Underlined amino acids were deter- mined by N-terminal sequence analysis from the purified enzyme. An asterisk shows the termination codon (TAA). The nucleotide from positions 1168 to 1176 was poly(A). The nucleotide sequence marked by a dotted line was identified as a genomic DNA sequence. Catalytic and noncatalytic zinc-binding residues are shown by arrows and arrowheads, respectively. The glycine- rich region (GXGXXG) corresponding to the NAD + -binding domain is in bold. K. Noge et al. Geraniol dehydrogenase from Carpoglyphus lactis FEBS Journal 275 (2008) 2807–2817 ª 2008 The Authors Journal compilation ª 2008 FEBS 2811 latipes ADH class VI. In the parsimony tree, Oryz- ias latipes ADH class VI was the sister group of the cluster consisting of all other animal ADHs. Discussion On the basis of differences in relative molecular mass, subunit composition, and cofactor requirement, Car. lactis GeDH should probably be classified sepa- rately from animal and plant ADHs, and plant GeD- Hs. Our results suggest that the purified Car. lactis GeDH may have a monomeric structure as an active enzyme. The discrepancy in relative molecular mass between the native and denatured forms might reflect the tertiary structure of the native enzyme. The calcu- lated relative molecular mass based on the deduced 378 amino acids of Car. lactis GeDH was 40 630, which is almost consistent with that determined by SDS ⁄ PAGE. The difference between the relative molecular masses of Car. lactis GeDH determined by SDS ⁄ PAGE and calculated from the deduced amino acid sequence may be due to the mobility of the enzyme on the gel or post-translational modification. ADHs thus far purified from plants [19,20], animals, including insects [21,22], and microorganisms [23,24] commonly function as dimers or oligomers. However, the subunit structure of Car. lactis GeDH was quite different from that of other ADHs. As far as we know, there is only one other example of a mono- meric ADH with activity, isolated from the Indian snakeroot, Rauwolfia serpentina [25]. The relative molecular mass of the purified dehydrogenase from Rau. serpentina is about 50 000 on the basis of gel fil- tration chromatography, and 44 000 by SDS ⁄ PAGE, and is thus comparable to that of Car. lactis GeDH. GeDHs have been partially purified from two plant sources, lemongrass [13] and orange [14]. The relative molecular masses of native GeDH from these materi- als are 84 000 and 92 000, respectively. Although the subunit composition of these GeDHs was not deter- mined, the relative molecular mass determined for native Car. lactis GeDH is smaller than that of either GeDH obtained from the above two species. More- over, the requirement for a cofactor (NADP + ) of the Fig. 6. Phylogenetic tree based on amino acid sequences of Car. lactis GeDH and other ADHs by minimum evolution criterion. The scale of the distances is shown under the tree. The tree is separated into three groups (group 1, ADHs from animals and plants; group 2, CADs and geraniol-related dehydrogenases; and group 3, Car. lactis GeDH). 10H-GeDH indicates 10-hydroxy- geraniol oxidoreductase. The numbers at the nodes represent the percentage of boot- strap confidence level (more than 50% is indicated). Geraniol dehydrogenase from Carpoglyphus lactis K. Noge et al. 2812 FEBS Journal 275 (2008) 2807–2817 ª 2008 The Authors Journal compilation ª 2008 FEBS GeDHs obtained from lemongrass [13], orange [14] and sweet basil [16] was also different from that of Car. lactis GeDH. The results from this study suggest that Car. lactis GeDH specializes in the biosynthesis of neral, the alarm pheromone of Car. lactis. This enzyme showed much greater specificity for the oxidation of geraniol than other alcohols tested (Table 2), and the properties of the enzyme were highly consistent with the biosyn- thetic pathway of neral in Car. lactis (Fig. 1). A previ- ous study has also shown that the oxidation of geraniol is one of the key steps in the production of neral [12]. The low amount of the enzyme may be due to its specific role in alarm pheromone biosynthesis. Citral, a mixture of neral and geranial, is known to have antifungal activity against As. fumigatus [10], Penicillium italicum, and two other pathogens [26]. Citral has also been reported to have a repellent effect on ants [27], which are possible predators in nature. These facts suggest that neral plays essential roles in the survival of Car. lactis. The high specificity of Car. lactis GeDH for geraniol might be driven by selective pressure to develop an ecologically advanta- geous character for the survival of this mite, efficient neral production. Car. lactis GeDH showed 50% oxi- dation activity with farnesol. Oxidation of farnesol is related to the biosynthesis of insect juvenile hormone [28,29]. As far as we know, there is no report of the presence of farnesal and its related compounds (e.g. juvenile hormones) in Car. lactis, so we cannot deter- mine the biological significance of farnesol oxidation by Car. lactis GeDH at this time. The pH range suitable for alcohol oxidation by Car. lactis GeDH is similar to that of other ADHs [22,30–32]. The highly conserved sequences of zinc-binding and NAD + -binding sites as compared to other ADHs suggest that Car. lactis GeDH belongs to the med- ium-chain dehydrogenase ⁄ reductase (MDR) family [33]. Comparison of the amino acid sequence of Car. lactis GeDH with that of other ADHs shows that seven residues implicated in the zinc-binding site are strictly conserved (Fig. 5). Three residues (Cys48, His75, and Cys179) and four cysteine residues (Cys105, Cys108, Cys111, and Cys119) in Car. lactis GeDH may serve as catalytic and noncatalytic zinc- binding sites, respectively. Our preliminary study showed that the geraniol oxidation activity of Car. lactis GeDH was inhibited by preincubation with 50 mm EDTA. However, the activity did not recover after addition of bivalent cation (Zn 2+ ,Cu 2+ ,Mn 2+ , or Co 2+ ) to the inactivated enzyme by EDTA treatment. Gel filtration chromatography of the inactivated enzyme showed an increase of apparent relative molecular mass as compared to the native enzyme (data not shown). It is thus possible that EDTA treatment leads to an irreversible structural change of Car. lactis GeDH that is coupled with its activity. Although the essential metal ion has not been identified, these results show that Car. lactis GeDH requires the metal ion for its oxidation activity and structural maintenance. The glycine-rich region (position 204–209, GLGGIG) is also conserved in Car. lactis GeDH (Fig. 5), and has been found in the NAD + -binding site in ADHs [34]. The absence of the probable initiator methionine in the native enzyme may be due to the N-terminal processing commonly observed in eukaryotic proteins [35]. Our phylogenetic analyses of the MDR family sug- gest that Car. lactis GeDH should be categorized as a new class of ADH. The primary structure of Car. lactis GeDH was more similar to animal ADHs (e.g. ostrich ADH class II and Ae. aegypti ADH) than to CADs and geraniol-related dehydrogenases (basil GeDH and two 10-hydroxygeraniol oxidoreductases) obtained from plants. The requirement for NAD + for oxidation was shared by other ADHs, whereas CADs and geraniol-related dehydrogenases require NADP + for their alcohol oxidation activity. On the other hand, the substrate specificity of Car. lactis GeDH was quite different from that of other ADHs, in that Car. lactis GeDH never oxidized ethanol. The capability for gera- niol oxidation was shared by basil CAD and GeDHs found in basil, lemongrass, orange, and ginger. The characteristics of Car. lactis GeDH seem to be a hybrid between those of animal ADHs and plant CADs in relation to primary structure and substrate specificity. In this article, we have reported that Car. lactis GeDH functions as a monomer, which is a unique sub- unit structure, and that this enzyme enables Car. lactis to produce neral efficiently. We also determined the primary structure of Car. lactis GeDH, which suggested that it should be categorized as a new class of ADHs, different from other ADHs, plant CADs, and plant GeDHs. Experimental procedures Mite Car. lactis Linnaeus (Acari: Carpoglyphidae) was originally of a strain maintained at Tokyo Women’s Medical College. The strain was maintained at 25 °C and 75% relative humidity, and fed a mixture of dry yeast and sugar (1 : 1) at the Laboratory of Chemical Ecology, Kyoto University, Japan. K. Noge et al. Geraniol dehydrogenase from Carpoglyphus lactis FEBS Journal 275 (2008) 2807–2817 ª 2008 The Authors Journal compilation ª 2008 FEBS 2813 Enzyme assay GeDH activity was assayed by monitoring the formation of NADH at 340 nm in a DU-64 spectrophotometer (Beck- man Coulter Inc., Fullerton, CA, USA) at room tempera- ture. Each assay was performed with 50 lL of reaction mixture containing 50 mm Tris ⁄ HCl (pH 8.5), 0.1 mm NAD + , 0.1 mm geraniol, and 1 lL of enzyme source. The reaction was started by adding l lL of the crude enzyme solution or partially purified fractions. One unit is defined as the amount of enzyme needed to generate 1 lmol NAD- HÆmin )1 . For determining the catalytic properties, 10 lgÆmL )1 purified enzyme was used in each assay. The protein concentration was determined by the Coomassie Blue method of Bradford [36], using BSA as a standard. To estimate the effect of temperature on the geraniol oxidation activity, the same reaction was performed at 4, 16, 25, 37 and 60 °C. After 30 min, the reaction mixture was extracted with the same quantity of hexane containing 5 p.p.m. octa- decane as an internal standard. The extract (3 lL) was subjected to GC and GC ⁄ MS analysis as previously described [37]. Each compound was identified by comparing its GC retention time and mass spectrum with those of authentic compounds. Enzyme purification All purification procedures were carried out at 4 °C unless otherwise stated. Mites (75 g) were separated from culture media by suspending them in saturated saline. Medium-free mites were left at 4 °C for more than 4 h. Initial experi- ments indicated that this cold storage resulted in maximal GeDH activity. Medium-free mites were then suspended in 50 mm Tris ⁄ HCl (pH 7.8) (buffer A) and homogenized using an ultrasonic processor, Astrason (Misonix Inc., Farmingdale, NY, USA). The homogenate was centrifuged (15 000 g, 30 min at 4 °C), and then the supernatant was recovered as an enzyme source. The 30–60% ammonium sulfate fraction was dissolved in 20% ammonium sulfate- saturated buffer A. The protein solution was loaded onto a TOYOPEARL Phenyl-650M column (2.0 · 18.6 cm; Tosoh Corp., Tokyo, Japan) and eluted with buffer A. A com- bined active fraction was loaded on a Toyoperal HW50-F column (2.4 · 12 cm; Tosoh Corp.) and eluted with buf- fer A. The active fractions were combined and loaded onto a DEAE–cellulose column (Whatman DE52, 1.5 · 6.0 cm; Whatman, Clifton, NJ, USA) and eluted with a gradient of 0–0.3 m NaCl in buffer A. A combined active fraction was loaded onto a Blue-Cellulofine column (1.1 · 3.0 cm; Seikagaku Corp., Tokyo, Japan) equilibrated with buffer A containing 0.5 m NaCl, and eluted with buffer A containing 2mm NAD + . The active fractions were combined and concentrated by ultrafiltration on a 10K Nanosep cen- trifugal device (Pall Corp., East Hills, NY, USA). The solu- tion was loaded onto a Superdex 200 HR 10 ⁄ 30 column (Amersham Biosciences, Uppsala, Sweden) and eluted with buffer A at 18 °C using an A ¨ KTA explorer 10S system (Amersham Biosciences). A combined active fraction was concentrated by ultrafiltration and loaded onto a MonoQ HR 5 ⁄ 5 column (Amersham Biosciences) at 18 °Conan A ¨ KTA explorer 10S system. After elution with a gradient of 0–0.3 m NaCl in buffer A, the active fractions were combined. A combined active fraction was desalted and then diluted to 10 lgÆmL )1 in buffer A for enzyme assay. Determination of relative molecular mass The apparent relative molecular mass of the purified Car. lactis GeDH was determined by gel filtration chroma- tography using a Superdex 75 HR 10 ⁄ 30 column (Amer- sham Biosciences) and by SDS ⁄ PAGE. The column was eluted with buffer A at 18 °C. The following proteins were used as M r standards: BSA (67 000), hen egg ovalbumin (43 000), bovine chymotrypsinogen A (25 000), and bovine ribonuclease A (13 700). SDS ⁄ PAGE was performed according to the procedure of Laemmli [38], using the following proteins as M r standards: rabbit phophorylase b (97 000), BSA (66 000), chicken egg ovalbumin (45 000), bovine carbonic anhydrase (30 000), soybean trypsin inhibi- tor (20 100), and bovine a-lactalbumin (14 400). N-terminal amino acid sequence analysis One hundred micrograms of purified Car. lactis GeDH from 351 g of fresh mites was transferred to a poly(vinyli- dene difluoride) membrane (0.2 lm; Bio-Rad, Hercules, CA, USA). Edman degradation was performed as described by Honda et al. [39]. RNA isolation and cDNA cloning of Car. lactis GeDH Total RNA was isolated from 1 g of Car. lactis within 30 min after separation from culture media according to Takano et al. [40]. Poly(A) + RNA was isolated using Oligotex-MAG (Takara, Kyoto, Japan) according to the manufacturer’s recommendation, and then was used for a 3¢-RACE reaction to synthesize first-strand cDNA using an adapter (3¢-Full RACE core set; Takara)-linked oligo (dT), 5¢-CTGATCTAGAGGTACCGGATCCTTTTTTTT TT(A ⁄ G ⁄ C)-3¢, and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manu- facturer’s recommendation. The cDNA was used directly for PCR amplification using the primer corresponding to an adapter as mentioned above and a specific degenerate primer, gdh-n1 (5¢-AARGARGGICARCCIATGAARATH GARCAR-3¢), designed on the basis of the partial N-termi- nal amino acid sequence of Car. lactis GeDH. PCR was performed in a 25 lL volume containing 0.63 units of Taq Geraniol dehydrogenase from Carpoglyphus lactis K. Noge et al. 2814 FEBS Journal 275 (2008) 2807–2817 ª 2008 The Authors Journal compilation ª 2008 FEBS DNA polymerase (Blend Taq; Toyobo, Osaka, Japan), 1· Blend Taq buffer, 0.2 mm dNTP, 0.4 lm each of the two primers, and 1 lL of the cDNA as a template. The PCR amplification was carried out as follows: an initial step of 2 min at 94 °C, followed by 30 cycles with denatur- ation at 94 °C for 30 s, annealing at 48 °C for 30 s, and extension at 72 °C for 2 min. After the final cycle, the products were extended for 5 min at 72 °C. An amplified product of  1.1 kbp was isolated by agarose gel electro- phoresis, purified, subcloned into the EcoRV site of the vector pZErO TM -2 (Invitrogen), and sequenced. DNA seq- uencing was performed as described in Yoshimi et al. [41]. Inverse PCR was performed with genomic DNA to obtain the 5¢-region of the nucleotide sequence encoding Car. lactis GeDH. Genomic DNA was isolated as described in Noge et al. [42], and was then digested with a restriction enzyme, HindIII (Takara), for 18 h at 37 °C. The digestions were followed by self-ligation using T4 DNA ligase (MBI Fermentas, St Leon-Rot, Germany). Inverse PCR was performed using Ex Taq DNA polymerase (Takara) with the self-ligated DNA as a template, and two primers, gdh-in-1c (5¢-CCAGACGCTCAAGGATGGCAAC-3¢) and gdh-in-1n (5¢-CTGGTGCCTGGATCAGGACCTG-3¢), designed on the basis of the cDNA sequence encoding Car. lactis GeDH. PCR parameters were 94 °C for 2 min and 30 cycles of 94 ° C for 30 s, 60 ° C for 40 s, and 72 °C for 4 min 30 s, followed by 72 °C for 5 min. Nested PCR was carried out using another pair of primers, gdh-in-1c and gdh-in-2n (5¢-GTGCCTGGATCAGGACCTGTTC-3¢), and the previous PCR products diluted by 1% as a template. The nested PCR reaction was performed with some modifications of the methods for inverse PCR; 35 cycles, and annealing at 57 °C for 40 s. The sequence of the resulting PCR product was determined as mentioned above. Phylogenetic analyses To evaluate the relationship of Car. lactis GeDH to the MDR family, the sequence data were selected on the basis of the similarity of amino acid sequence and substrate specific- ity to Car. lactis GeDH. Amino acid sequences correspond- ing to several classes of ADH found in a wide range of species (animals, including insects, plants, and micro- organisms) were obtained from UniProtKB ⁄ Swiss-Prot and UniProtKB ⁄ TrEMBL. Their origins and primary accession numbers are as follows: human, Homo sapiens (ADH1A, P07327; ADH1B, P00325; ADH1C, P00326; ADH4, P08319; ADH5, P11766; ADH6, P28332; ADH7, P40394); Norway rat, Rat. norvegicus (ADH4, Q64563; ADH class III, P12711; class IV, P41682); ostrich, St. camelus (ADH1, P80338; ADH class II, P80468); zebrafish, Brachydanio rerio (ADH5, Q6NXA6); Japanese medaka, Oryzias latipes (ADH class III, Q6R5I9; class VI, Q6B4J3); African clawed frog, Xenopus laevis (ADH5, Q6EE34); Florida lancelet, Branchiostoma floridae (ADH class III, Q9BBJ34); fruit fly, Drosophila melanogaster (ADH class III, P46415); yellow fever mosquito, Ae. aegypti (Q176A7); African malaria mosquito, Anopheles gambiae (Q7Q8X4); rice, Oryza sativa (ADH1, P20306; ADH2, P18332); potato, So. tuberosum (ADH1, P14673; ADH2, P14674; ADH3, P14675); tomato, So. lycopersicum (ADH2, P28032); baker’s yeast, Saccharo- myces cerevisiae (YADH1, P00330; YADH2, P00331; YADH5, P38113); and Neurospora crassa (Q9P6C8). The N-terminal sequences showed similarity to plant ADHs (see Results), so they were also used for phylogenetic analyses. ADHs found in microorganisms were used as outgroups. CAD found in sweet basil, Oc. basilicum (Q2KNL5), and tobacco, Nicotiana tabacum (P30359), GeDH found in Oc. basilicum (Q2KNL6), and 10-hydroxygeraniol oxidore- ductase found in Camptotheca acuminata (Q7XAB2), and the Madagascar periwinkle, Catharanthus roseus (Q6V4H0), were also used. Of the enzymes related to geraniol oxidation, the only sequences available are of basil GeDH and basil CAD [16]. Although there is no evidence for the ability of tobacco CAD to oxidize geraniol, it was used as an example of a CAD to clarify whether Car. lactis GeDH belonged to the same cluster of plant CADs. Two sequences from Cam. acuminata and Cat. roseus are annotated as 10-hydroxygeraniol oxidoreductases, although their bio- chemical characteristics have not yet been reported. These two sequences show high similarity to basil GeDH [16]. Amino acid sequences were aligned using the clustal w (v. 1.81) multiple alignment program [43] with its default parameters, and then the aligned sequences were checked manually. The phylogenetic tree was inferred using maxi- mum parsimony and minimum evolution (distance) criteria with the paup* program version 4.0b10, written by D. L. Swofford [44]. Maximum parsimony analysis was performed using the heuristic search option with stepwise addition of 10 random replicates and tree-bisection- reconnection branch-swapping. For the minimum evolution analysis, mean character difference was used as the distance measure, and tree-bisection-reconnection branch-swapping was applied to the starting tree, obtained via the neighbor- joining algorithm. Gaps are treated as missing for both analyses. Bootstrap values [45] were obtained from 1000 replicate resampled datasets for both methods. Acknowledgements We thank Dr J. X. 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Analysis and Using Parsimony (and other methods) Sinauer Associates Inc., Sunderland, MA 45 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap Evolution 39, 783–791 FEBS Journal 275 (2008) 2807–2817 ª 2008 The Authors Journal compilation ª 2008 FEBS 2817 . Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae) Koji. found in the NAD + -binding site in ADHs [34]. The absence of the probable initiator methionine in the native enzyme may be due to the N-terminal processing