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Characterization of Thermophilic Archaeal Isopentenyl Phosphate Kinases

机译：嗜热古代戊烯基激酶的表征

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摘要

Archaea synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the essential building blocks of isoprenoid compounds, from mevalonate (MVA). However, an analysis of the genomes of several members of the Archaea failed to identify genes for the enzymes required to convert phosphomevalonate (PM) to IPP in eukaryotes. The recent discovery of an isopentenyl kinase (IPK) in Methanocaldococcus jannaschii (MJ) suggests a new variation of the MVA pathway where PM is decarboxylated to give isopentenyl phosphate (IP), which is phosphorylated to produce IPP. A blast search using the MJ protein as a probe revealed a subfamily of amino acid kinases that include the fosfomycin resistance protein fomA, which deactivates the antibiotic by phosphorylation of its phosphonate residue in a reaction similar to the conversion of IP to IPP. IPK genes were cloned from two organisms identified in the search, Methanothermobacter thermautotrophicus (MTH) and Thermoplasma acidophilum (THA), and the His-tagged recombinant proteins were purified by Ni-NTA chromatography. The enzymes catalyze the reversible phosphorylation of IP by ATP, K_(eq)=6.3 ± 1. The catalytic efficiencies (V/K) of the proteins were ～2 × 10~6M~(-1) s~(-1). In the reverse direction, ADP was a substrate inhibitor for THAIPK, K_i~(ADP)=58 ± 6 μM, but not forMTHIPK. Both enzymes were active over a broad range of pH and temperature. Five compounds, dimethylallyl phosphate, isopentenyl thiolophosphate, 1-butyl phosphate, 3-buten-1-yl phosphate, and geranyl phosphate, were evaluated as alternative substrates for the MTH and THA IP kinases. All of the compounds were phosphorylated, although the catalytic efficiency was low for geranyl phosphate.
机译：Archaea合成异戊基二磷酸二磷酸（IPP）和二甲基allyL二磷酸（DMAPP），来自甲戊酯（MVA）的异戊二烯化合物的基本结构块。然而，对古痤疮的几个成员的基因组的分析未能鉴定在真核生物中转化磷脂素（PM）的酶所需的基因。最近在甲基甲烷基团jannaschii（MJ）中的异戊烯基激酶（IPK）的发现表明了MVA途径的新变化，其中PM是脱羧，得到异戊基磷酸酯（IP），其磷酸化以产生IPP。使用MJ蛋白作为探针的爆炸搜索揭示了氨基酸激酶的亚家族，其包括氟霉素抗性蛋白质FOMA，其通过其膦酸盐残基的磷酸化与IP转化为IPP的反应中的磷酸化残基磷酸化失活。从搜索中鉴定的两种生物中克隆了IPK基因，并通过Ni-NTA色谱法纯化了他标记的重组蛋白。酶催化ATP的可逆磷酸化IP，K_（EQ）= 6.3±1.蛋白质的催化效率（v / k）为约2×10〜6m〜（-1）S〜（-1）。在相反的方向上，ADP是THAIPK的底物抑制剂，K_I〜（ADP）= 58±6μm，但不是骨球率。两种酶在广泛的pH和温度范围内都是活性的。评价了五种化合物，二甲基丙烯酸磷酸，异戊烯基，三丁酯，3-丁烯-1-基磷酸磷酸盐和磷酸甲苯烷基酯，作为MTH和THA IP激酶的替代底物。所有化合物都磷酸化，但催化效率低对磷酸甲酯低。

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