Báo cáo khoa học: Identification and biochemical characterization of the Anopheles gambiae 3-hydroxykynurenine transaminase pot

Identification and biochemical characterization of the Anopheles gambiae 3-hydroxykynurenine transaminase Franca Rossi 1 , Fabrizio Lombardo 2 , Alessandra Paglino 1 , Camilla Cassani 1 , Gianluca Miglio 1 , Bruno Arca ` 2,3 and Menico Rizzi 1 1 DiSCAFF, University of Piemonte Orientale ‘Amedeo Avogadro’, Novara, Italy 2 Dipartimento di Scienze di Sanita ` Pubblica – Sezione di Parassitologia, Universita ` di Roma ‘La Sapienza’, Italy 3 Dipartimento di Biologia Strutturale e Funzionale, Universita ` di Napoli ‘Federico II’, Italy In the last decades, the catabolic intermediates arising from the main pathway for tryptophan oxidative de- gradation, collectively known as kynurenines, have received great attention on account of their roles in the physiological tuning of the central nervous system and in the etiogenesis and progression of several human neurodegenerative diseases (reviewed in [1–4]). One of the most powerful cytotoxic metabolites of the kynure- nine pathway is the 3-hydroxykynurenine (3-HK), which is generated in the third step of the catabolic cascade, through the hydroxylation of the central com- mon precursor l-kynurenine (L-KYN) [5–7]. Sponta- neous oxidation of the 3-HK induces free radical generation and apoptosis as shown both in vitro and in animal models [8,9]. Mammals can scavenge the poten- tially toxic 3-HK excess either by converting 3-HK to xanthurenic acid (XA) by direct transamination or by hydrolysing the molecule to produce alanine and 3-hydroxyanthranilic acid (3-HAA); this metabolic intermediate is then fluxed into the central branch of the catabolic cascade, resulting in the de novo synthesis of the essential cofactor NAD [10]. 3-Hydroxykynure- nine detoxification is a metabolic priority also for the vast majority of other living species that depend on Keywords Anopheles gambiae; 3-hydroxykynurenine transaminase; xanthurenic acid; Plasmodium gametogenesis; PLP dependent enzyme Correspondence M. Rizzi, DiSCAFF, University of Piemonte Orientale ‘Amedeo Avogadro’, Via Bovio 6, 28100 Novara, Italy Fax: +39 0321375821 Tel: +39 0321375812 E-mail: menico.rizzi@pharm.unipmn.it (Received 26 July 2005, revised 31 August 2005, accepted 6 September 2005) doi:10.1111/j.1742-4658.2005.04961.x Spontaneous oxidation of 3-hydroxykynureine (3-HK), a metabolic inter- mediate of the tryptophan degradation pathway, elicits a remarkable oxida- tive stress response in animal tissues. In the yellow fever mosquito Aedes aegypti the excess of this toxic metabolic intermediate is efficiently removed by a specific 3-HK transaminase, which converts 3-HK into the more sta- ble compound xanthurenic acid. In anopheline mosquitoes transmitting malaria, xanthurenic acid plays an important role in Plasmodium gameto- cyte maturation and fertility. Using the sequence information provided by the Anopheles gambiae genome and available ESTs, we adopted a PCR- based approach to isolate a 3-HK transaminase coding sequence from the main human malaria vector A. gambiae. Tissue and developmental expres- sion analysis revealed an almost ubiquitary profile, which is in agreement with the physiological role of the enzyme in mosquito development and 3-HK detoxification. A high yield procedure for the expression and purifi- cation of a fully active recombinant version of the protein has been devel- oped. Recombinant A. gambiae 3-HK transaminase is a dimeric pyridoxal 5¢-phosphate dependent enzyme, showing an optimum pH of 7.8 and a comparable catalytic efficiency for both 3-HK and its immediate catabolic precursor kynurenine. This study may be useful for the identification of 3-HK transaminase inhibitors of potential interest as malaria transmission- blocking drugs or effective insecticides. Abbreviations 3-HAA, 3-hydroxyanthranilic acid; 3-HK, 3-hydroxykynurenine; 3-HKT, specific 3-hydroxykynurenine transaminase; KA, kynurenic acid; KAT, kynurenine aminotransferase; L-KYN, L-kynurenine; PLP, pyridoxal 5¢-phosphate; UTR, untranslated region; XA, xanthurenic acid. FEBS Journal 272 (2005) 5653–5662 ª 2005 FEBS 5653 the kynurenine pathway for dietary tryptophan oxida- tion and that are sensitive to the potential toxic effects of its intermediate metabolites. Indeed, in adult insects of different species, exogenously administered kynure- nines can have a deep impact on complex behaviours [11] and locomotive activity [12,13]; this impairment can culminate with the irreversible paralysis of the insect and death [14]. Interestingly, it has been shown that 3-HK can produce its effects by a direct killing of neurons, through a well characterized series of cyto- pathological events which have several traits in com- mon with the rat striatum neuron apoptotic death induced by 3-HK treatment [15]. So far a specific 3-hydroxykynurenine transaminase (3-HKT) function has not been identified in man. In fact, in mammals 3-HK can undergo two alternative fates: it can be hydrolysed to 3-HAA through the action of a specific kynureninase [16] or, as alternative, it can be transformed to XA by the same pyridoxal 5¢-phosphate (PLP)-dependent kynurenine amino- transferase (KAT) isozymes that catalyse the irrevers- ible transamination of L-KYN to kynurenic acid (KA) [17–19]. In the yellow fever mosquito Aedes aegypti, a kynu- reninase enzymatic activity has never been documented; therefore, the detoxification of 3-HK should rely totally on the capability of the insect to transform 3-HK into the more stable XA. Initially it was assumed that A. aegypti KAT [20] could sustain both L-KYN and 3-HK transamination in the mosquito, similar to what is observed for the human enzyme. However, this hypo- thesis has been recently disproved by the observation that A. aegypti KAT has no activity towards 3-HK and by the discovery of a specific 3-HKT enzyme (Ae-HKT ⁄ AGT), catalysing the irreversible transamina- tion of 3-HK to XA [21,22]. An increasing number of molecular and developmental studies highlighted the essential role of XA in the gametogenesis and fertility of the malaria parasite [23,24]. Very recently, the molecu- lar details of the signalling cascade triggered by XA and resulting in the maturation of Plasmodium gametes have been elucidated [25]. Overall, the available data point to XA metabolism as a likely crucial crossroad in the bio- logy of Plasmodium development into the mosquito. However, very little is known about how anophelines deal with metabolic 3-HK excess. In particular, the bio- chemical aspects of the 3-HK dependent synthesis of XA in Anopheles gambiae, the most efficient vector of the most deadly malaria parasite P. falciparum, remain elusive. We report here the identification, the bacterial heterologous expression and the biochemical characteri- zation of 3-HKT from A. gambiae. These results repre- sent the first biochemical report concerning an enzyme involved in the kynurenine pathway in A. gambiae. Our analysis on 3-HKT contributes to a better understand- ing of the XA metabolism in the mosquito and offers a new possible target for the development of insecticides and ⁄ or transmission blocking agents. Results and Discussion Isolation of the A. gambiae 3-HKT cDNA In order to isolate the A. gambiae 3-HKT cDNA, the sequence of the Aedes aegypti enzyme (Ae-HKT ⁄ AGT, accession number AF435806) [21] was used to search the A. gambiae genome by the blast search tool [26,27]. A genomic region whose conceptual translation showed high level of similarity to the Aedes aegypti enzyme was retrieved; this sequence was used to search the EST database allowing for the identification of two nonoverlapping ESTs probably representing the 5¢-end (BM637288) and the 3¢-end (BM603618) of the 3-HKT A. gambiae cDNA. Oligonucleotide primers matching these ESTs were used to amplify by RT-PCR the puta- tive coding region of the A. gambiae 3-HKT cDNA. This strategy allowed for the isolation of a 1400-bp long cDNA clone (accession number AM042695); according to the expected function of the encoded protein, we named the corresponding gene Ag-hkt. Sequence analysis revealed that the Ag-hkt cDNA clone contains a single 1188-bp long ORF (position 174–1361 in Fig. 1) flanked by two short untranslated regions (UTRs). The conceptual translation of this ORF resulted in a 396-amino acid long protein, repre- senting the A. gambiae 3-HKT. Expression profile of the 3-HKT gene The expression of the Ag-hkt gene was analysed by RT-PCR amplification of the corresponding mRNA from different A. gambiae developmental stages and from a few selected adult tissues. As shown in Fig. 2, Ag-hkt is expressed at significantly high levels through- out embryonic ⁄ larval development and in both adult males and females, whereas expression is very low or absent during the pupal stage. This developmental pat- tern of expression is on line with earlier observations made in A. aegypti and with the physiological need of the mosquito to keep 3-HK under stringent control [21,28]. Indeed, during embryo and larval development and in mosquito adulthood, the expression of Ag-hkt is important in protecting insect tissues from the 3-HK-triggered oxidative stress response. On the other hand, downregulation of Ag-hkt expression in pupae is in agreement with the physiological 3-HK requirement A. gambiae 3-hydroxykynurenine transaminase F. Rossi et al. 5654 FEBS Journal 272 (2005) 5653–5662 ª 2005 FEBS for the production of ommochromes during compound eye development [29,30]. Moreover, in vivo develop- mental studies suggest that 3-HK may play an active role in tissue remodelling during neurometamorphosis by induction of free radical generations and apoptosis [31]. As far as tissue-specific expression is concerned, high levels of Ag-hkt expression were found in ovaries and in the gut (Fig. 2, lane 6 and 7). These observa- tions fit well both with the previously reported expres- sion in the developing ovaries of Aedes aegypti [21] as well as with the involvement of the enzyme in the oxi- dative degradation of dietary tryptophan. Moreover, a recent microarray analysis includes the Ag-hkt among a group of genes that are upregulated upon blood- feeding in A. gambiae [32]. The very low or almost undetectable expression in the salivary glands (Fig. 2, lane 5) is somehow unexpected because XA has been found in A. stephensi saliva [33]; indeed, it has been suggested that salivary glands may represent an important source of XA which would be delivered into the host with saliva during blood-feeding and then ingested along with blood into the midgut [33]. The results of our tissue-specific expression analysis are in agreement with a recent study on the A. gambiae tran- scriptome that failed to reveal Ag-hkt mRNA among over 850 salivary transcripts [34]. Therefore, we suggest that the involvement of salivary glands in the supply of the gametocyte activating factor XA may be of limited importance in comparison to that of the midgut. Overall the observed Ag-hkt expression profile closely resembles the one reported for the Ae-HKT ⁄ AGT [21] and points to the mosquito 3-HKT as a possible novel target for the development of insecticides which, by targeting both the larval and adult stages of 5'- tgttacggtagcggtacctgttt gccgaagtgttgtcagcttggtgttctagagtgagggtataactaacgctgccctaaagttggaagaaggggaataacgtaaacgacaca cctcagtgacattgtgcgaattgtcccgtattgtattaacttactgaaagtgctgatacaatgaagttcacgccgccccctgcatcgcta MKFTPPPASL cgcaatcctttaatcattccggaaaagataatgatgggccctggaccgtccaactgctcaaagcgggtgctgactgccatgactaacacc RNPLIIPEKIMMGPGPSNCSKRVLTAMTNT gtgctgagcaacttccacgctgaattgttccgaacgatggacgaggtcaaggatggcttgcggtacatttttcagacagaaaaccgggcc VLSNFHAELFRTMDEVKDGLRYIFQTENRA actatgtgcgtaagcggttccgcacacgcgggaatggaagctatgctgagcaatctgcttgaagagggcgatcgagtgctgatcgcggtt TMCVSGSAHAGMEAMLSNLLEEGDRVLIAV aacggaatttgggcagagcgtgccgtcgaaatgtctgagcgatacggtgccgatgttcgaacgattgagggccctccggaccgcccgttc NGIWAERAVEMSERYGADVRTIEGPPDRPF agtttggaaacattggccagagccatcgaactgcatcaacccaagtgtctgttcctgacgcacggtgactcatcaagtggtctgctgcag SLETLARAIELHQPKCLFLTHGDSSSGLLQ ccgctggaaggtgtaggccagatttgtcaccagcacgactgtttgctcatcgttgatgccgtggcttcgctgtgcggtgtgccattttat PLEGVGQICHQHDCLLIVDAVASLCGVPFY atggataaatgggagattgatgccgtctatactggagcgcaaaaggtgctaggtgcgcctcctggtatcactcccatttctataagcccg MDKWEIDAVYTGAQKVLGAPPGITPISISP aaagcactggatgttattcgaaatcgtcgtacaaaatcgaaagtattttactgggatttactgctgcttggcaattattggggctgttat KALDVIRNRRTKSKVFYWDLLLLGNYWGCY gatgaaccaaaacgttatcaccatactgtcgcatcgaacttaatatttgctctgcgggaagcattggctcaaattgcggaagaaggactg DEPKRYHHTVASNLIFALREALAQIAEEGL gaaaatcagatcaaacgccgcatcgaatgtgcccaaatcttgtacgaagggcttggtaagatgggactcgatattttcgtgaaagacccc ENQIKRRIECAQILYEGLGKMGLDIFVKDP agacatcgcctgcccaccgtaactggtattatgattccgaaaggtgttgactggtggaaagtttcacaatacgccatgaacaatttttcg RHRLPTVTGIMIPKGVDWWKVSQYAMNNFS ttagaagtacaaggaggacttggacctacgtttggaaaagcatggcgtgtgggtattatgggcgaatgctcaacggtacaaaaaatacaa LEVQGGLGPTFGKAWRVGIMGECSTVQKIQ ttctatctatatggctttaaggaatcactcaaagccacgcatcccgactatattttcgaggaaagtaatggatttcactagacgaaactt FYLYGFKESLKATHPDYIFEESNGFH aaacaatgcatcaatgtattattgccg - 3' Fig. 1. Sequence analysis of the A. gambiae 3-HKT. The full length sequence of the isolated Ag-hkt cDNA clone: the putative translation start codon and stop codon are shown in bold. The primary sequence of the conceptual translation product Ag-HKT is shown. 1 234 56789 ag-hk t rpS7 Fig. 2. Developmental- and tissue-specific Ag-hkt expression analysis. RT-PCR amplification of total RNA from different tissues and develop- mental stages with Ag-hkt- and rpS7 -specific primers. Lanes: 1, embryos; 2, larvae; 3, pupae; 4, adult females; 5, salivary glands; 6, ovaries; 7, midgut; 8, carcasses (adult females with salivary gland, ovaries and gut removed); 9, adult males. F. Rossi et al. A. gambiae 3-hydroxykynurenine transaminase FEBS Journal 272 (2005) 5653–5662 ª 2005 FEBS 5655 the mosquito, would represent a valuable tool for fighting mosquito-borne diseases by vector control. Recombinant Ag-HKT bacterial expression and enzyme purification To obtain large amounts of highly purified A. gambiae 3-HKT for subsequent enzymatic studies, we expressed the corresponding coding sequence in a bacterial high- producing system. To this end, we subcloned the PCR amplified Ag-hkt orf into the NcoI ⁄ XhoI site of the pET-16b plasmid, thus eliminating the vector region encoding the standard poly-histidine tract. The result- ing pET ⁄ Ag-hkt construct drives, in Escherichia coli, the T7-dependent expression of a no-tags bearing, 396- amino acid long recombinant version of the mosquito enzyme (Ag-HKT). The SDS ⁄ PAGE analysis (Fig. 3, lane A) reveals that the recombinant Ag-HKT (predic- ted relative molecular mass, 44 298 Da) represents the major band in the soluble fraction of a lysate from pET ⁄ Ag-hkt transformed E. coli BL21(DE3) cells. The starting relative abundance of the recombinant Ag-HKT ( 50% of soluble proteins), together with an intrinsic robustness of the enzyme, allowed us to adopt a simple protocol for its purification to homo- geneity, consisting of a preliminary ammonium sul- phate fractionation followed by three FPLC steps exploiting anion exchange media displaying increasing separation power. This purification procedure repro- ducibly yielded  8 mg of 99% pure recombinant Ag-HKTÆL )1 of transformed bacterial culture (Fig. 3, lane B). The purified protein was yellow in colour and aUV⁄ VIS spectroscopic analysis (Fig. 3, panel C) revealed the classic spectral profile for a PLP-bound aminotransferase, where the peak at 410 nm corres- ponds to the internal aldimine form of the cofactor [35,36]. The absorption spectra at different pH values ranging from 6.0 to 9.0 were also measured. Differ- ently to what was observed in other PLP-dependent enzymes, such as aspartate aminotransferase and 1-aminocyclopropane-1-carboxylate synthase [37], Ag-HKT does not exhibit a pH dependent spectrum of its internal aldimine (data not shown). Moreover, the recombinant Ag-HKT was able to catalyse 3-HK transamination (see hereafter), definit- ively confirming that we had isolated a full-length cDNA clone encoding a functional protein. Size exclu- sion chromatography performed on the pure and act- ive enzyme revealed a dimeric quaternary assembly for the recombinant protein (data not shown). Table 1 summarizes the 3-HKT specific activity recovered after each purification step. Substrate specificity and kinetic analysis of the recombinant Ag-HKT Enzymatic assays demonstrated that Ag-HKT cata- lyses the transamination of both 3-HK and kynurenine to XA and KA, respectively, using different a-keto- acids as amino group acceptor cosubstrates. Among MWM (kDa) BA 29 45 66 116 97 Abs 300 350 400 450 500 550 600 0.000 0.010 0.020 0.030 0.040 0.050 0.060 0.070 0.080 0.090 0.100 nm C 410 nm Fig. 3. Analysis of the bacterial expressed and purified Ag-HKT. Analysis by SDS ⁄ PAGE of the protein content of the pET ⁄ Ag-hkt trans- formed E. coli BL21(DE3) clarified lysate (A) and of the purified recombinant enzyme (B); samples were separated on a 10% polyacrylamide gel and stained by Coomassie blue. The arrowhead points to the overexpressed Ag-HKT and to the recovered enzyme at the end of the puri- fication procedure. MWM: relative molecular mass markers (kDa). (C) Absorption spectrum of the internal aldimine of a 10 l M solution of Ag-HKT in 200 m M potassium phosphate (pH 7.0) at 25 °C. The arrowhead indicates to the 410 nm maximum absorption peak. A. gambiae 3-hydroxykynurenine transaminase F. Rossi et al. 5656 FEBS Journal 272 (2005) 5653–5662 ª 2005 FEBS these, the maximal activity towards both 3-HK and L-KYN was observed with glyoxylate; pyruvate effi- ciently substituted for glyoxylate only in the Ag-HKT catalysed transamination of 3-HK, whereas oxaloace- tate and a-ketobutyrate acted as less efficient amino acceptors with both substrates (Table 2). Conse- quently, the determination of the kinetic parameters of the Ag-HKT catalysed reactions was invariably per- formed using glyoxylate as the cosubstrate. Because pure L-3-HK is not available from any com- mercial source, all of the data presented refer to experi- ments performed by using a racemic mixture of l- and d-3-HK (d,l-3HK in Tables 2 and 3). To compare the catalytic parameters featuring Ag-HKT activity towards l-3-hydroxykynurenine and l-kynurenine, we performed the kinetic analysis also by using d,l-KYN, a racemic mixture of kynurenine equivalent to that used in the case of 3-HK. The kinetic parameters deter- mined in the case of d,l-3HK and d,l-KYN (Tables 2 and 3) allowed us to conclude that A. gambiae 3-HKT displays comparable affinity and catalytic efficiency for the two substrates, as previously reported for the Aedes aegypti enzyme [21,22]. Neither the substrates nor the products of the Ag-HKT-dependent reactions exerted any inhibitory effect on the catalysis, which follows a classical Michaelis–Menten kinetics. Moreover, we tes- ted the effect on the 3-HK transamination catalysed by Ag-3HKT, of all the natural amino acids at a concen- tration of 1 mm without observing any enzyme inhibi- tion (data not shown). The concentration we used in these experiments greatly exceeds the one present in a typical mosquito blood meal [38]. Therefore, the Ag- HKT capability of sustaining the necessary synthesis of XA in the mosquito midgut is not expected to be signi- ficantly influenced by other amino acids present in the blood meal. The effect of pH and temperature on Ag-HKT catalysed synthesis of XA We examined next the pH optimum and temperature dependence of the 3-HK transamination catalysed by the purified recombinant Ag-HKT. Of functional rele- vance, the enzyme possesses a pH optimum of 7.8 (Fig. 4A); this value is close to the pH range of 7.5– 7.7 recorded in the midgut of A. stephensi females after blood feeding [39]. Interestingly, although Ag-3HKT and Aa-3HKT show a sequence identity of 79%, a sharp difference in their optimum pH is observed, with the Aedes aegypti enzyme displaying an optimum pH of 9.0 [21]. Such an observation suggests that fine tuning of A. gambiae 3-HKT activity is likely to be instrumental Table 1. Recombinant Ag-HKT purification procedure. The starting amount of soluble proteins in the lysate form pET ⁄ ag-hkt trans- formed bacteria and the proteins content after each purification step were quantified by Bradford assays. The specific transamina- tion activity towards 3-HK of each purification sample was deter- mined as described in Experimental procedures using glyoxylate as the amino acceptor substrate. Purification step Total protein (mg) Specific activity (lmolÆmin )1 Æmg )1 ) Total activity (lmolÆmin )1 ) Activity yield (%) Lysate (soluble fraction) 594.4 10.3 6122.3 100 Ammonium sulfate precipitation 272 11.7 3182.4 52 HiTrap Q 109 17.7 1929.3 31.5 Source Q 28 26.3 736.4 12 Mono Q 13.4 32.6 436.8 7 Table 2. Ag-HKT specificity towards different amino acceptors. Data are expressed as percentage of the activity obtained using glyoxylate as the a-ketoacid amino acceptor in the Ag-HKT cata- lysed transamination of D,L-3HK or L-KYN as detailed in Experimen- tal procedures. Cosubstrate Amino acid substrate Glyoxylate Pyruvate Oxalacetate a-Ketobutyrate D,L-3HK 100 96.8 55.4 52.7 L-KYN 100 59.5 62.1 41.2 Table 3. Kinetic parameters for Ag-HKT. Kinetic parameters of the transamination reactions of Ag-HKT towards D,L-3HK, D,L-KYN, L-KYN were determined by using glyoxylate as the a-ketoacid amino acceptor, whereas the corresponding values for glyoxylate were determined by using D,L-3HK as the amino donor. Each assay has been performed in triplicate. Substrate K m (mM) V max (lmolÆmin )1 ) k cat (min )1 ) k cat ⁄ K m (min )1 ÆmM )1 ) D,L-3HK 2.0 ± 0.3 0.044 ± 0.005 988.5 ± 113 494.3 ± 80 D,L-KYN 2.3 ± 0.3 0.048 ± 0.013 1077.2 ± 284 468.3 ± 44 L-KYN 1.0 ± 0.4 0.028 ± 0.009 611.5 ± 214 611.5 ± 9 Glyoxylate 2.7 ± 0.1 0.102 ± 0.003 2264.8 ± 67.9 838.8 ± 48.9 F. Rossi et al. A. gambiae 3-hydroxykynurenine transaminase FEBS Journal 272 (2005) 5653–5662 ª 2005 FEBS 5657 to an efficient synthesis of XA in mosquito midgut. The curve of the enzymatic activity as a function of tem- perature, revealed that Ag-HKT is highly thermostable and works well in a wide temperatures interval, ranging from 25° to 65 °C, with a maximum activity peak, in our in vitro tests, at 60 °C (Fig. 4B). However, it should be noted that at  30 °C, i.e. at temperatures close to physiological values, the recombinant enzyme still works at 60–65% of its maximum, with an activity of  20 lmolÆmin )1 Æmg )1 . Of note is that in the same con- ditions, the A. aegypti homologue functions at  20% of its maximum [21], with an activity that is four times lower than that of the A. gambiae enzyme ( 5 lmolÆ min )1 Æmg )1 ). The possible physiological ⁄ functional significance and the implications of these differences between the Aedes aegypti and the A. gambiae enzymes are presently unclear; however, it should be noted that P. gallinaceum, a parasite species that is transmitted by Aedes aegypti, requires in vitro XA concentration of 80 nm for the induction of gametogenesis at half- maximal response (EC 50 ), whereas P. falciparum and P. berghei, that are transmitted by anopheline mosqui- toes, require concentrations of XA that are of 2 lm and 9 lm, respectively, i.e. 25–100 times higher [40]. Overall, we have shown here that the A. gambiae Ag-hkt gene is expressed at relatively high level in the midgut and that the encoded 3-HKT enzyme acting on the 3-HK produced by the oxidative degradation of the tryptophan after blood feeding, may represent the major source of mosquito-derived XA, a key inducer of gametogenesis and a crucial player in the initiation of parasite development into the mosquito. Conclusions Plasmodium male gametocytes full maturation, thr- ough the spectacular phenomenon of exflagellation [41], requires the presence of XA which is needed in vitro in the micromolar range for most Plasmo- dium species [40]. In principle, XA may potentially derive from the tryptophan metabolism of any of the three organisms involved in malaria: the vector, the vertebrate host and the parasite. However, the para- site itself does not give any contribution and the average XA concentration in human blood has been determined to be in the 0.6–2 lm range [42], a value that would theoretically support exflagellation of P. falciparum at 13–50% maximal efficiency [40], sug- gesting that endogenously mosquito-produced XA may be of key importance for sustaining exflagella- tion. Interestingly, a putative 3-HKT gene has recently been shown to be highly upregulated in A. gambiae adult females fed with P. bergheii infected blood [43]. Within such a scenario, the isolation and biochemical characterization of recombinant A. gambiae 3-HKT that we report here, represents a step for- ward into the understanding of XA metabolism in Fig. 4. The effect of pH and temperature on Ag-HKT-mediated cata- lysis. (A) The optimum pH for Ag-HKT was monitored following a 5-min incubation of the samples at 50 °C. In each test, D,L-3HK and glyoxylate were used as the amino donor and acceptor, respect- ively; the reaction mixtures were prepared in buffer phosphate at 5.5, 6.5, 7.4, 7.8, 8.0, 8.5, 9.0 pH values. (B) The temperature dependence of Ag-HKT catalysed reaction was monitored at either pH 7.8 (filled squares) or pH 7.0 (open squares) by incubating the corresponding samples 5 min at 23, 37, 50, 60, 80, 100 °C. Each assay was initiated by the addition of 2 lg of the purified enzyme to the prewarmed reaction mixtures. All experiments were per- formed in triplicate. A. gambiae 3-hydroxykynurenine transaminase F. Rossi et al. 5658 FEBS Journal 272 (2005) 5653–5662 ª 2005 FEBS A. gambiae and will be useful for the identification of enzyme inhibitors of potential interest as malaria transmission-blocking drugs and⁄ or effective insecti- cides. Experimental procedures Mosquito colony The A. gambiae used in this study is the GASUA reference strain (Xag, 2R, 2La, 3R, 3 L, SuaKoKo, Liberia) main- tained under standard conditions. The different tissues and developmental stages were collected, immediately frozen in liquid nitrogen and stored at )80 °C until used for RNA extraction. Tissues were dissected in a few drops of NaCl ⁄ P i and stored in ice for no longer than 60 min before freezing. 3-HKT cDNA cloning and expression analysis Nucleic acid manipulations, if not otherwise specified, were performed according to standard procedures [26] or follow- ing manufacturers’ instructions. Total RNA was extracted from the different tissues and developmental stages using the TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA). Approximately 80 ng DNase I-treated RNA (RNase-free DNase I, Invitrogen) extracted from 2–4-day old adult females were used for the cDNA amplification by the SuperScript one-step RT-PCR system (Invitrogen). Reverse transcription (50 °C, 30 min) and heat inactivation of the reverse transcriptase (94 °C, 2 min) were followed by 35 cycles of amplification (30 s, 94 °C; 30 s, 58 ° C; 90 s, 72 °C) and a final elongation of 7 min at 72 °C. The ampli- fication product was digested with BamHI and EcoRI, gel purified, cloned into the pBCKSII vector (Stratagene, La Jolla, CA, USA) and sequenced. Forward and reverse prim- ers, containing at their ends, respectively, BamHI and EcoRI restriction sites, were designed using the sequence information from the two nonoverlapping ESTs BM637288 and BM603618 representing the putative 5¢-UTR and 3¢-UTR of the A. gambiae 3-HKT. The sequence of the oligonucleotide primers was: hkt1BamF, 5¢-GATCGGATC CTGTTACGGTAGCGGTACCTG-3¢; hkt1EcoR, 5¢-CAT GGAATTCCGGCAATAATACATTGATGC-3¢. For the expression analysis approximately 80–100 ng of DNase I-treated total RNA were amplified by the Super- Script one-step RT-PCR system using the following Ag-hkt and rpS7 (ribosomal protein S7) oligonucleotide primers: hktF, 5¢-TGTAGGCCAGATTTGTCACC-3¢,hktR.5¢-CCTT CTTCCGCAATTTGAGC-3¢; rpS7F, 5¢-GGCGATCATCA TCTACGTGC-3¢; rpS7R, 5¢-GTAGCTGCTGCAAACTT CGG-3¢. Briefly, reverse transcription (50 °C, 30 min) and heat inactivation of the reverse transcriptase (94 °C, 2 min) were followed by 35 cycles of amplification (30 s, 94 °C; 30 s, 55 °C; 60 s, 72 °C) and a final elongation of 7 min at 72 °C. Twenty-five cycles were used for the amplification of rpS7 mRNA in order to keep the reaction below saturation and to allow a more reliable normalization. The different stages used for expression analysis were: embryos, 0–48 h; larvae, first to fourth instar larvae; pupae, early and late pupae; 2–4 day-old adult females; 2–4 day-old adult males. Tissues were dissected from adult females 2–4 days after emergence. Carcasses were adult females without salivary glands, ovaries and gut. Recombinant bacterial expression vector construction The 1191-bp Ag-HKT coding sequence, from the putative translational ATG to the end of the ORF, was amplified by PCR using the corresponding A. gambiae cDNA clone (see above) as the template. The PCR primers used were: for_3hkt, 5¢-TTAACCATGGTGAAGTTCACGCCGCCCC CT-3¢; rev_3hkt, 5¢-TTAACTCGAGTCTAGTGAAATCCA TTACTTTCCTC-3¢ and they were designed to contain, respectively, the NcoI and the XhoI restriction sites at their 5¢ ends (in italic). The NcoI–XhoI digested PCR product was ligated into NcoI–XhoI linearized pET16b vector (Novagen, Madison, WI, USA), resulting in the pET ⁄ Ag-hkt construct. Ag-HKT bacterial expression and recombinant enzyme purification Several colonies of E. coli BL21(DE3) freshly transformed with the pET ⁄ ag-hkt construct, were inoculated in 1 litre of Luria–Bertani medium (50 lgÆmL )1 ampicillin), and grown at 22 °C under vigorous shaking. At 18 h postinoculation, bacteria were collected by centrifugation (11 000 g for 15 min at 4 °C) and resuspended in 40 mL lysis solution consisting of 100 mm phosphate buffer, pH 6.8, 40 lm PLP and protease inhibitor cocktail. After ultrasonication on ice, the bacterial lysate was clarified by centrifugation (14 000 g for 45 min at 4 °C) and ammonium sulphate was added to the resulting supernatant (20% saturation). Preci- pitated proteins were removed by low speed centrifugation (10 000 g for 30 min at 4 °C) and the soluble material was further fractionated in 50% saturated ammonium. The pre- cipitate was collected as above, resuspended in 15 mL puri- fication buffer (20 mm Tris ⁄ HCl pH 8.0, 40 lm PLP and proteases inhibitor cocktail) and extensively dialysed. Recombinant Ag-HKT was purified by FPLC chromato- graphy (Akta Basic instrument, Amersham Pharmacia Biosciences, Milan, Italy) using, in sequence, HiTrapQ, SourceQ and MonoQ prepacked anion exchange media (Amersham Pharmacia Biosciences). A linear 0.0–0.5 m NaCl gradient was invariably used to elute retained pro- teins. Flow-through protein content was monitored by a double wavelength reading at 280 nm (total proteins) and F. Rossi et al. A. gambiae 3-hydroxykynurenine transaminase FEBS Journal 272 (2005) 5653–5662 ª 2005 FEBS 5659 at 425 nm (internal aldimine form of the PLP cofactor). Cromatographic fractions showing absorbance at 425 nm were analysed by standard SDS ⁄ PAGE. Pure Ag-HKT containing fractions from the last chromatographic step were pooled, dialysed against 20 mm Tris ⁄ HCl pH 8.0, 150 mm NaCl and concentrated by ultrafiltration using a 30 000 MWCO disposable device. The Ag-HKT concentra- tion was determined by a Bradford assay, using BSA as the standard. The purity of the recombinant enzyme was assessed by standard SDS ⁄ PAGE analysis. UV ⁄ visible absorption spectra of the purified recombinant Ag-HKT were measured by using a UV ⁄ VIS spectrometer at room temperature. A 10-lm enzyme solution was scanned within the 300–650 nm wavelength interval. Aliquots of Ag-HKT, supplemented by 40 lm PLP and proteases inhibitor cock- tail, were stored at 4 ° C (up to 1 week storage) or at )80 °C (long-term storage). Recombinant Ag-HKT biochemical characterization The activity of bacterial expressed Ag-HKT was assayed by measuring the amount of XA produced following the incuba- tion of the enzyme with d,l-3HK as amino group donor in the presence of different a-ketoacids as amino group accep- tors, according to literature [21,22]. Two micrograms of recombinant Ag-HKT were incubated at 50 °C in 200 mm potassium phosphate buffer pH 7.0, 40 lm PLP and 5 mm d,l-3HK in presence of either 5 mm glyoxylate, pyruvate, a-ketobutyrate or oxalacetate, in a final reaction volume of 50 lL. Each reaction was started by the addition of the enzyme and stopped after 5 min by adding an equal volume of formic acid (0.8 m). The precipitated enzyme was removed by centrifugation at 16 000 g for 10 min at 4 °C and a 20-lL aliquot of the resulting supernatants was injected into a HPLC-UV system (System Gold, Beckman Coulter, Milan, Italy) equipped with a C-18 sphereclone ODS [2] analytical column (5 lm particle size, 250 · 4.0 mm; Phenomenex, Torrance, CA, USA). The mobile phase (50 mm potassium phosphate buffer, pH 4.8, 10% v ⁄ v acetonitrile) was deliv- ered at a flow-rate of 1 mLÆ min )1 at room temperature and the XA formation was monitored by UV absorbance meas- urement at 330 nm. Identification of each compound in the assay mixture was based on its specific retention time and coelution with each standard. To quantify the XA produced in each experiment, a calibration curve was constructed using the same HPLC-UV experimental setting. To check the ami- notransferase activity of the recombinant Ag-HKT towards kynurenine, an identical approach was followed, substituting either l-KYN or d,l-KYN for 3-HK as amino group donors and fixing at 5 mm the amino group acceptor glyoxylate (see Results). The synthesis of the transamination product KA was monitored at 330 nm and quantified by interpolating the experimental data in the corresponding standard curve obtained as above. The kinetic parameters of Ag-HKT catalysis towards d,l-3HK, l-KYN and d,l-KYN were determined under the same experimental conditions described above, by adding increasing concentrations of amino donors (0.3–5 mm) into the corresponding reaction mixtures. Similarly, the K m and V max values of the Ag-HKT reaction towards the amino acceptor cosubstrate glyoxylate, were obtained by adding increasing concentrations of the molecule to the reaction samples containing 5 mmd,l-3HK as the amino donor. The amount of XA produced after a 5-min incubation was measured and data were fitted to the Michaelis–Menten equation. Effect of pH and temperature on the Ag-HKT activity The Ag-HKT pH optimum was determined by adding 2 lg of the recombinant enzyme to each reaction mixture con- taining 200 m m phosphate buffer at different pH values (Fig. 4A legend), 5 mmd,l-3HK, 5 mm glyoxylate and 40 lm PLP; samples were incubated for 5 min at 50 °C. The temperature dependence of Ag-HKT activity towards 3-HK was similarly studied by incubating the samples (5 mmd,l-3HK, 5 mm glyoxylate, 40 lm PLP in 200 mm phosphate buffer either pH 7.0 or pH 7.8) for 5 min at the indicated temperature values (Fig. 4B legend). In both cases, the specific activity of the enzyme was determined by quantifying the XA formed by HPLC-UV analysis. 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In particular, the bio- chemical aspects of the 3-HK dependent synthesis of XA in Anopheles gambiae, the most efficient vector of the most deadly malaria