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

Identification and biochemical characterization of the Anopheles gambiae 3-hydroxykynurenine transaminase Franca Rossi1, Fabrizio Lombardo2, Alessandra Paglino1, Camilla Cassani1, Gianluca Miglio1, Bruno Arca` 2,3 and Menico Rizzi1

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

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.

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- the 3-hydroxykynurenine (3-HK), nine pathway is 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-

the 3-HK induces free radical neous oxidation of 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

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.

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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

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].

(PLP)-dependent

kynurenine

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 it can be hydrolysed to 3-HAA through the fates: action of a specific kynureninase [16] or, as alternative, it can be transformed to XA by the same pyridoxal 5¢-phosphate amino- transferase (KAT) isozymes that catalyse the irrevers- ible transamination of L-KYN to kynurenic acid (KA) [17–19].

translation of

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. the Ag-hkt cDNA Sequence analysis revealed that clone contains a single 1188-bp long ORF (position 174–1361 in Fig. 1) flanked by two short untranslated regions (UTRs). The conceptual this ORF resulted in a 396-amino acid long protein, repre- senting the A. gambiae 3-HKT.

Expression profile of the 3-HKT gene

in protecting insect

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 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

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

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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.

for the production of ommochromes during compound in vivo develop- eye development [29,30]. Moreover, 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 M K F T P P P A S L cgcaatcctttaatcattccggaaaagataatgatgggccctggaccgtccaactgctcaaagcgggtgctgactgccatgactaacacc R N P L I I P E K I M M G P G P S N C S K R V L T A M T N T gtgctgagcaacttccacgctgaattgttccgaacgatggacgaggtcaaggatggcttgcggtacatttttcagacagaaaaccgggcc V L S N F H A E L F R T M D E V K D G L R Y I F Q T E N R A actatgtgcgtaagcggttccgcacacgcgggaatggaagctatgctgagcaatctgcttgaagagggcgatcgagtgctgatcgcggtt T M C V S G S A H A G M E A M L S N L L E E G D R V L I A V aacggaatttgggcagagcgtgccgtcgaaatgtctgagcgatacggtgccgatgttcgaacgattgagggccctccggaccgcccgttc N G I W A E R A V E M S E R Y G A D V R T I E G P P D R P F agtttggaaacattggccagagccatcgaactgcatcaacccaagtgtctgttcctgacgcacggtgactcatcaagtggtctgctgcag S L E T L A R A I E L H Q P K C L F L T H G D S S S G L L Q ccgctggaaggtgtaggccagatttgtcaccagcacgactgtttgctcatcgttgatgccgtggcttcgctgtgcggtgtgccattttat P L E G V G Q I C H Q H D C L L I V D A V A S L C G V P F Y atggataaatgggagattgatgccgtctatactggagcgcaaaaggtgctaggtgcgcctcctggtatcactcccatttctataagcccg M D K W E I D A V Y T G A Q K V L G A P P G I T P I S I S P aaagcactggatgttattcgaaatcgtcgtacaaaatcgaaagtattttactgggatttactgctgcttggcaattattggggctgttat K A L D V I R N R R T K S K V F Y W D L L L L G N Y W G C Y gatgaaccaaaacgttatcaccatactgtcgcatcgaacttaatatttgctctgcgggaagcattggctcaaattgcggaagaaggactg D E P K R Y H H T V A S N L I F A L R E A L A Q I A E E G L gaaaatcagatcaaacgccgcatcgaatgtgcccaaatcttgtacgaagggcttggtaagatgggactcgatattttcgtgaaagacccc E N Q I K R R I E C A Q I L Y E G L G K M G L D I F V K D P agacatcgcctgcccaccgtaactggtattatgattccgaaaggtgttgactggtggaaagtttcacaatacgccatgaacaatttttcg R H R L P T V T G I M I P K G V D W W K V S Q Y A M N N F S ttagaagtacaaggaggacttggacctacgtttggaaaagcatggcgtgtgggtattatgggcgaatgctcaacggtacaaaaaatacaa L E V Q G G L G P T F G K A W R V G I M G E C S T V Q K I Q ttctatctatatggctttaaggaatcactcaaagccacgcatcccgactatattttcgaggaaagtaatggatttcactagacgaaactt Y F K L Y G E S L A T H P D Y I F E E S N G F H K F aaacaatgcatcaatgtattattgccg - 3'

1

2

3

4

5

6

7

8

9

ag-hkt

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.

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A. gambiae 3-hydroxykynurenine transaminase

for

the mosquito, would represent a valuable tool fighting mosquito-borne diseases by vector control.

Recombinant Ag-HKT bacterial expression and enzyme purification

synthase

Ag-HKTÆL)1 of transformed bacterial culture (Fig. 3, lane B). The purified protein was yellow in colour and a UV ⁄ 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 such as aspartate aminotransferase and enzymes, 1-aminocyclopropane-1-carboxylate [37], Ag-HKT does not exhibit a pH dependent spectrum of its internal aldimine (data not shown).

relative

the

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

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 recombinant abundance of starting Ag-HKT ((cid:1) 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 (cid:1) 8 mg of 99% pure recombinant

A

B

MWM (kDa)

C 0.100

0.090

0.080

410 nm

116 97

0.070

66

0.060

Abs

0.050

45

0.040

0.030

0.020

29

0.010

0.000

300

350

400

450

500

550

600

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 lM solution of Ag-HKT in 200 mM potassium phosphate (pH 7.0) at 25 (cid:1)C. The arrowhead indicates to the 410 nm maximum absorption peak.

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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)

Activity yield (%)

Specific activity (lmolÆmin)1Æmg)1)

Total activity (lmolÆmin)1)

Lysate (soluble

594.4

10.3

6122.3

100

272

11.7

3182.4

52

fraction) Ammonium

sulfate precipitation

109 28 13.4

17.7 26.3 32.6

1929.3 736.4 436.8

31.5 12 7

HiTrap Q Source Q Mono Q

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.

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.

The effect of pH and temperature on Ag-HKT catalysed synthesis of XA

Because pure L-3-HK is not available from any com- mercial source, all of the data presented refer to experi-

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.

Amino acid substrate

Glyoxylate Pyruvate Oxalacetate

Cosubstrate

a-Ketobutyrate

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].

D,L-3HK L-KYN

100 100

96.8 59.5

55.4 62.1

52.7 41.2

Such an observation suggests that fine tuning of A. gambiae 3-HKT activity is likely to be instrumental

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

)1)

Km (mM)

Vmax (lmolÆmin)1)

kcat (min)1)

kcat ⁄ Km (min)1ÆmM

D,L-3HK D,L-KYN L-KYN Glyoxylate

2.0 ± 0.3 2.3 ± 0.3 1.0 ± 0.4 2.7 ± 0.1

0.044 ± 0.005 0.048 ± 0.013 0.028 ± 0.009 0.102 ± 0.003

988.5 ± 113 1077.2 ± 284 611.5 ± 214 2264.8 ± 67.9

494.3 ± 80 468.3 ± 44 611.5 ± 9 838.8 ± 48.9

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from 25(cid:1) to 65 (cid:1)C, with a maximum activity peak, in our in vitro tests, at 60 (cid:1)C (Fig. 4B). However, it should be noted that at (cid:1) 30 (cid:1)C, i.e. at temperatures close to physiological values, the recombinant enzyme still works at 60–65% of its maximum, with an activity of (cid:1) 20 lmolÆmin)1Æmg)1. Of note is that in the same con- ditions, the A. aegypti homologue functions at (cid:1) 20% of its maximum [21], with an activity that is four times lower than that of the A. gambiae enzyme ((cid:1) 5 lmolÆ min)1Æmg)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 (EC50), 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

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 (cid:1)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 (B) The temperature 5.5, 6.5, 7.4, 7.8, 8.0, 8.5, 9.0 pH values. 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 (cid:1)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.

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

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 exflagellation of that would theoretically support P. falciparum at 13–50% maximal efficiency [40], sug- gesting that endogenously mosquito-produced XA may be of key importance for sustaining exflagella- Interestingly, a putative 3-HKT gene has tion. 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

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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

30 s, 55 (cid:1)C; 60 s, 72 (cid:1)C) and a final elongation of 7 min at 72 (cid:1)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 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 (cid:1)C until used for RNA extraction. Tissues were dissected in a few drops of NaCl ⁄ Pi and stored in ice for no longer than 60 min before freezing.

3-HKT cDNA cloning and expression analysis

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

the bacterial

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 (cid:1)C, 30 min) and heat inactivation of the reverse transcriptase (94 (cid:1)C, 2 min) were followed by 35 cycles of amplification (30 s, 94 (cid:1)C; 30 s, 58 (cid:1)C; 90 s, 72 (cid:1)C) and a final elongation of 7 min at 72 (cid:1)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- their ends, respectively, BamHI and ers, containing at 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¢.

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 (cid:1)C under vigorous shaking. At 18 h postinoculation, bacteria were collected by centrifugation (11 000 g for 15 min at 4 (cid:1)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, lysate was clarified by centrifugation (14 000 g for 45 min at 4 (cid:1)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 (cid:1)C) and the soluble material was further fractionated in 50% saturated ammonium. The pre- cipitate was collected as above, resuspended in 15 mL puri- (20 mm Tris ⁄ HCl pH 8.0, 40 lm PLP fication buffer 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

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 (cid:1)C, 30 min) and heat inactivation of the reverse transcriptase (94 (cid:1)C, 2 min) were followed by 35 cycles of amplification (30 s, 94 (cid:1)C;

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the

experimental

catalysis towards d,l-3HK, l-KYN and d,l-KYN were determined under conditions same described above, by adding increasing concentrations of amino donors (0.3–5 mm) into the corresponding reaction mixtures. Similarly, the Km and Vmax 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 mm d,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

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 the recombinant enzyme was standard. The purity of 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 (cid:1)C (up to 1 week storage) or at )80 (cid:1)C (long-term storage).

Recombinant Ag-HKT biochemical characterization

The Ag-HKT pH optimum was determined by adding 2 lg of the recombinant enzyme to each reaction mixture con- taining 200 mm phosphate buffer at different pH values (Fig. 4A legend), 5 mm d,l-3HK, 5 mm glyoxylate and 40 lm PLP; samples were incubated for 5 min at 50 (cid:1)C. The temperature dependence of Ag-HKT activity towards 3-HK was similarly studied by incubating the samples (5 mm d,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.

Acknowledgements

This work was supported by grants from MIUR (FIRB2001), Regione Piemonte (Ricerca sanitaria finalizzata 2004) and Fondazione Cariplo (project number 2004.1591 ⁄ 11.5437) to M. R. and by the EU BioMalPar NoE n. 503578 to B. A. and Mario Col- uzzi. A. P. is the recipient of a fellowship from Regi- one Piemonte (Ricerca scientifica applicata, 2003).

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