7,8-Diaminoperlargonic acid aminotransferase from Mycobacterium tuberculosis, a potential therapeutic target
7,8-Diaminoperlargonic acid aminotransferase from Mycobacterium tuberculosis, a potential therapeutic target
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DOI：
10.1111/j.1742-4658.2006.05479.x
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发表时间：
2006-10-01
期刊：
影响因子：
5.4
通讯作者：
Ploux, Olivier
中科院分区：
文献类型：
--
作者：
Mann, Stephane;Ploux, Olivier
参与生物素生物合成的二氨基苯丙酸氨基转移酶(DAPAAT)以S-腺苷-L-蛋氨酸(ADOMet)为氨基供体，催化8-氨基-7-氧农酸(KAPA)的转氨化反应。结核分枝杆菌DAPA AT，一个潜在的治疗靶点，已在大肠杆菌中大量生产，并通过镍亲和层析的单一有效步骤纯化到均一。该酶表现出典型的吡哆醛5‘-磷酸依赖酶的电子吸收光谱，在溶液中表现为同源四聚体。饱和时活性的pH谱显示为单一电离基团，其pK(A)为8.0，这归因于活性中心赖氨酸残基。该酶具有较强的底物抑制作用，其动力学参数为：K-mADOMet=0.78+/-0.20 mM，K-Mkapa=3.8+/-1.0 mU M，k(CAT)=1.0+/-0.2 min(-1)，K-iKAPA=14+/-2 mU M。阿米克霉素和新类似物4-(4C-氨基环六-2，5-二烯-1R-基)丙醇(简称化合物1)是该酶的自杀底物。阿米克霉素和化合物1的失活参数分别为：K-i=12+/-2muM，k(Inact)=0.35+/-0.05min(-1)，K-i=20+/-2muM，k(Inact)=0.56+/-0.05min(-1)。这种失活是不可逆的，阿米克霉素和化合物1的分配比分别为1.0和1.1，这使得这些失活剂特别有效。化合物1(100微克·毫升(-1))可完全抑制结核分枝杆菌BIOA基因(编码DAPA AT)转化大肠杆菌C268 bioA突变株的生长。加入生物素或DAPA可逆转其抗菌作用。因此，化合物1在体内特异性靶向DAPA AT。
Diaminopelargonic acid aminotransferase (DAPA AT), which is involved in biotin biosynthesis, catalyzes the transamination of 8-amino-7-oxononanoic acid (KAPA) using S-adenosyl-L-methionine (AdoMet) as amino donor. Mycobacterium tuberculosis DAPA AT, a potential therapeutic target, has been overproduced in Escherichia coli and purified to homogeneity using a single efficient step on a nickel-affinity column. The enzyme shows an electronic absorption spectrum typical of pyridoxal 5'-phosphate-dependent enzymes and behaves as a homotetramer in solution. The pH profile of the activity at saturation shows a single ionization group with a pK(a) of 8.0, which was attributed to the active-site lysine residue. The enzyme shows a Ping Pong Bi Bi kinetic mechanism with strong substrate inhibition with the following parameters: K-mAdoMet = 0.78 +/- 0.20 mM, K-mKAPA = 3.8 +/- 1.0 mu M, k(cat) = 1.0 +/- 0.2 min(-1), K-iKAPA = 14 +/- 2 mu M. Amiclenomycin and a new analogue, 4-(4c-aminocyclohexa-2,5-dien-1r-yl)propanol (referred to as compound 1), were shown to be suicide substrates of this enzyme, with the following inactivation parameters: K-i = 12 +/- 2 mu M, k(inact) = 0.35 +/- 0.05 min(-1), and K-i = 20 +/- 2 mu M, k(inact) = 0.56 +/- 0.05 min(-1), for amiclenomycin and compound 1, respectively. The inactivation was irreversible, and the partition ratios were 1.0 and 1.1 for amiclenomycin and compound 1, respectively, which make these inactivators particularly efficient. compound 1 (100 mu g.mL(-1)) completely inhibited the growth of an E. coli C268bioA mutant strain transformed with a plasmid expressing the M. tuberculosis bioA gene, coding for DAPA AT. Reversal of the antibiotic effect was observed on the addition of biotin or DAPA. Thus, compound 1 specifically targets DAPA AT in vivo.