Rahlfs Stefan, Koncarevic Sasa, Iozef Rimma, Mailu Boniface Mwongela, Savvides Savvas N, Schirmer R Heiner, Becker Katja
Interdisciplinary Research Center, Giessen University, Giessen, Germany.
Mol Biochem Parasitol. 2009 Feb;163(2):77-84. doi: 10.1016/j.molbiopara.2008.09.008. Epub 2008 Oct 8.
Adenylate kinases (AK; ATP+AMP<-->2 ADP; E.C. 2.7.4.3.) are enzymes essentially involved in energy metabolism and macromolecular biosynthesis. As we reported previously, the malarial parasite Plasmodium falciparum possesses one genuine AK and one GTP-AMP phosphotransferase. Analysis of the P. falciparum genome suggested the presence of one additional adenylate kinase, which we designated AK2. Recombinantly produced AK2 was found to be a monomeric protein of 33 kDa showing a specific activity of 10 U/mg with ATP and AMP as a substrate pair and to interact with the AK-specific inhibitor P(1),P(5)-(diadenosine-5')-pentaphosphate (IC(50)=200 nM). At its N-terminus AK2 carries a predicted myristoylation sequence. This sequence is only present in AK2 of P. falciparum causing the severe tropical malaria and not in other malarial parasites. We heterologously coexpressed AK2 and P. falciparum N-myristoyltransferase (NMT) in the presence of myristate in Escherichia coli. As demonstrated by protein purification and mass spectrometry, AK2 is indeed myristoylated under catalysis of the parasites' transferase. The modification significantly enhances the stability of the kinase. Furthermore, AK2 and NMT were shown to interact strongly with each other forming a heterodimeric protein in vitro. To our knowledge this is the first direct evidence that P. falciparum NMT myristoylates an intact malarial protein.
腺苷酸激酶（AK；ATP + AMP⇌2 ADP；E.C. 2.7.4.3.）是主要参与能量代谢和大分子生物合成的酶。正如我们之前报道的，疟原虫恶性疟原虫拥有一种真正的AK和一种GTP - AMP磷酸转移酶。对恶性疟原虫基因组的分析表明存在另一种腺苷酸激酶，我们将其命名为AK2。发现重组产生的AK2是一种33 kDa的单体蛋白，以ATP和AMP作为底物对时具有10 U/mg的比活性，并与AK特异性抑制剂P(1),P(5)-(二腺苷-5')-五磷酸相互作用（IC(50)=200 nM）。AK2在其N端带有一个预测的肉豆蔻酰化序列。该序列仅存在于导致严重热带疟疾的恶性疟原虫的AK2中，而不存在于其他疟原虫中。我们在大肠杆菌中在肉豆蔻酸存在的情况下异源共表达AK2和恶性疟原虫N - 肉豆蔻酰转移酶（NMT）。通过蛋白质纯化和质谱分析表明，在寄生虫转移酶的催化下AK2确实被肉豆蔻酰化。这种修饰显著增强了激酶的稳定性。此外，AK2和NMT在体外被证明彼此强烈相互作用形成异二聚体蛋白。据我们所知，这是恶性疟原虫NMT对完整疟原虫蛋白进行肉豆蔻酰化的首个直接证据。
