gambiae 3-HKT cDNA, the sequence of the Aedes aegypti enzyme Ae-HKT⁄ AGT, accession number AF435806 [21] was used to search the A.. The result-ing pET⁄ Ag-hkt construct drives, in Escher
Trang 1Identification and biochemical characterization of the
Anopheles gambiae 3-hydroxykynurenine transaminase Franca Rossi1, Fabrizio Lombardo2, Alessandra Paglino1, Camilla Cassani1, Gianluca Miglio1, Bruno Arca`2,3and 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
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-hydroxykynurekynure-nine (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 aegyptithe 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.
Trang 2the 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
Trang 3for 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
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
F Y L Y G F K E S L K A T H P D Y I F E E S N G F H
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.
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.
Trang 4the 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
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 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) A B
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.
Trang 5these, 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 concentrainhibi-tion 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 purificatransamina-tion sample was
deter-mined as described in Experimental procedures using glyoxylate as
the amino acceptor substrate.
Purification
step
Total
protein
(mg)
Specific activity (lmolÆmin)1Æmg)1)
Total activity (lmolÆmin)1)
Activity yield (%) Lysate (soluble
fraction)
Ammonium
sulfate
precipitation
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
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 Km(m M ) Vmax(lmolÆmin)1) kcat(min)1) kcat⁄ K m (min)1Æm M )1)
Trang 6to 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 60C (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)1Æmg)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)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
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.
Trang 7A 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⁄ Piand 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 (50C, 30 min) and heat inactivation of the
reverse transcriptase (94C, 2 min) were followed by 35
cycles of amplification (30 s, 94C; 30 s, 58 C; 90 s,
72C) 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 (94C, 2 min)
were followed by 35 cycles of amplification (30 s, 94C;
30 s, 55C; 60 s, 72 C) and a final elongation of 7 min at
72C Twenty-five cycles were used for the amplification of rpS7mRNA 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 22C under vigorous shaking At 18 h postinoculation, bacteria were collected by centrifugation (11 000 g for
15 min at 4C) 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 4C) 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 4C) 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
Trang 8at 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 4C (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 50C 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 4C 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 mLÆmin)1at 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 Kmand Vmaxvalues 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
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 50C 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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