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Characterization of Thermophilic Archaeal Isopentenyl Phosphate Kinases

机译：嗜热古细菌异戊烯基磷酸激酶的表征

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Archaea synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), thenessential building blocks of isoprenoid compounds, from mevalonate (MVA). However, an analysis of thengenomes of several members of the Archaea failed to identify genes for the enzymes required to convertnphosphomevalonate (PM) to IPP in eukaryotes. The recent discovery of an isopentenyl kinase (IPK) innMethanocaldococcus jannaschii (MJ) suggests a new variation of the MVA pathway where PM isndecarboxylated to give isopentenyl phosphate (IP), which is phosphorylated to produce IPP. A blast searchnusing the MJ protein as a probe revealed a subfamily of amino acid kinases that include the fosfomycinnresistance protein fomA, which deactivates the antibiotic by phosphorylation of its phosphonate residue in anreaction similar to the conversion of IP to IPP. IPK genes were cloned from two organisms identified in thensearch, Methanothermobacter thermautotrophicus (MTH) and Thermoplasma acidophilum (THA), and thenHis-tagged recombinant proteins were purified by Ni-NTA chromatography. The enzymes catalyze thenreversible phosphorylation of IP by ATP, Keq =6.3 ( 1. The catalytic efficiencies (V/K) of the proteins weren∼2u0001 106nM-1nsn-1n. In the reverse direction, ADP was a substrate inhibitor for THA IPK, KinADPn=58 (6 μM,nbut not forMTHIPK. Both enzymeswere active over a broad range of pHand temperature. Five compounds,ndimethylallyl phosphate, isopentenyl thiolophosphate, 1-butyl phosphate, 3-buten-1-yl phosphate, andngeranyl phosphate, were evaluated as alternative substrates for the MTH and THA IP kinases. All of thencompounds were phosphorylated, although the catalytic efficiency was low for geranyl phosphate
机译：古细菌由甲羟戊酸酯（MVA）合成异戊二烯化合物的重要结构单元异戊烯基二磷酸酯（IPP）和二甲基烯丙基二磷酸酯（DMAPP）。但是，对古细菌中几个成员的基因组进行的分析未能确定真核生物中将戊戊酸戊酸酯（PM）转化为IPP所需酶的基因。詹氏甲烷球菌（MJ）中的异戊烯基激酶（IPK）的最新发现表明MVA途径的新变化，其中PM未脱羧生成磷酸异戊烯基（IP），后者被磷酸化生成IPP。以MJ蛋白为探针进行的原始搜索揭示了一个氨基酸激酶的亚家族，其中包括磷霉素抗性蛋白fomA，它通过磷酸化其膦酸酯残基而使抗生素失活，其反应类似于IP向IPP的转化。从随后研究中鉴定的两种生物中克隆了IPK基因，即嗜热甲烷亚生菌（MTH）和嗜酸嗜热菌（THA），然后通过Ni-NTA色谱法纯化了带有His标签的重组蛋白。这些酶催化ATP的IP可逆磷酸化，Keq = 6.3（1.蛋白的催化效率（V / K）为n〜2u0001 106nM-1nsn-1n。相反，ADP是THA IPK的底物抑制剂， KinADPn = 58（6μM，对MTHIPK无效）。两种酶均在很宽的pH和温度范围内具有活性。评估了五种化合物，正磷酸二甲基烯丙酯，异戊烯基硫代磷酸酯，磷酸1-丁酯，磷酸3-丁烯酯和磷酸戊烯酯作为MTH和THA IP激酶的替代底物，所有化合物均被磷酸化，尽管磷酸香叶酯的催化效率较低

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  • 来源
    《Biochemistry》 |2010年第1期|p.207-217|共11页
  • 作者

    Mo Chen and C. Dale Poulter;

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    Department of Chemistry, University of Utah, Salt Lake City, Utah 84112 Present address: Department of Pharmacology,University of Pennsylvania School of Medicine, 135 John Morgan Building, 3620 Hamilton Walk,Philadelphia, PA 19104-6084;

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