Bathke Jochen, Fritz-Wolf Karin, Brandstädter Christina, Burkhardt Anja, Jortzik Esther, Rahlfs Stefan, Becker Katja
Biochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University, D-35392 Giessen, Germany.
Biochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University, D-35392 Giessen, Germany; Max-Planck Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany.
J Mol Biol. 2016 Dec 4;428(24 Pt B):4946-4961. doi: 10.1016/j.jmb.2016.10.023. Epub 2016 Oct 27.
Nicotinic acid mononucleotide adenylyltransferase (NaMNAT) is an indispensable enzyme for the synthesis of NAD and NAD phosphate. It catalyzes the adenylylation of nicotinic acid mononucleotide (NaMN) to yield nicotinic acid adenine dinucleotide (NaAD). Since NAD(H) and NAD phosphate(H) are essentially involved in metabolic and redox regulatory reactions, NaMNAT is an attractive drug target in the fight against bacterial and parasitic infections. Notably, NaMNAT of the malaria parasite Plasmodium falciparum possesses only 20% sequence identity with the homologous human enzyme. Here, we present for the first time the two X-ray structures of P. falciparum NaMNAT (PfNaMNAT)-in the product-bound state with NaAD and complexed with an α,β-non-hydrolizable ATP analog-the structures were determined to a resolution of 2.2Å and 2.5Å, respectively. The overall architecture of PfNaMNAT was found to be more similar to its bacterial homologs than its human counterparts although the PPHK motif conserved in bacteria is missing. Furthermore, PfNaMNAT possesses two cysteine residues within the active site that have not been described for any other NaMNATase so far and are likely to be involved in redox regulation of PfNaMNAT activity. Enzymatic studies and surface plasmon resonance data reveal that PfNaMNAT is capable of utilizing NaMN and nicotinamide mononucleotide with a slight preference for NaMN. Surprisingly, a comparison with the active site of Escherichia coli NaMNAT showed very similar architectures, despite different substrate preferences.
烟酰胺单核苷酸腺苷酸转移酶（NaMNAT）是合成NAD和NAD磷酸必不可少的酶。它催化烟酰胺单核苷酸（NaMN）的腺苷酸化反应生成烟酸腺嘌呤二核苷酸（NaAD）。由于NAD（H）和NAD磷酸（H）主要参与代谢和氧化还原调节反应，因此NaMNAT是对抗细菌和寄生虫感染的一个有吸引力的药物靶点。值得注意的是，恶性疟原虫的NaMNAT与同源的人类酶的序列同一性仅为20%。在此，我们首次展示了恶性疟原虫NaMNAT（PfNaMNAT）处于与产物NaAD结合状态以及与α,β-非水解性ATP类似物复合状态下的两个X射线晶体结构，其分辨率分别为2.2Å和2.5Å。尽管细菌中保守的PPHK基序不存在，但发现PfNaMNAT的整体结构与其细菌同源物比与其人类对应物更相似。此外，PfNaMNAT在活性位点内有两个半胱氨酸残基，这是迄今任何其他NaMNAT酶都未描述过的，并且可能参与PfNaMNAT活性的氧化还原调节。酶学研究和表面等离子体共振数据表明，PfNaMNAT能够利用NaMN和烟酰胺单核苷酸，对NaMN略有偏好。令人惊讶的是，尽管底物偏好不同，但与大肠杆菌NaMNAT的活性位点比较显示出非常相似的结构。
