Mutation of archaeal isopentenyl phosphate kinase highlights mechanism and guides phosphorylation of additional isoprenoid monophosphates.
Mutation of archaeal isopentenyl phosphate kinase highlights mechanism and guides phosphorylation of additional isoprenoid monophosphates.
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DOI：
10.1021/cb1000313
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发表时间：
2010-06-18
影响因子：
4
通讯作者：
Noel, Joseph P.
中科院分区：
文献类型：
--
作者：
Dellas, Nikki;Noel, Joseph P.
异戊烯基二磷酸（IPP）的生物合成无论是从甲羟戊酸（MVA）还是从1-脱氧-d-木酮糖5-磷酸（DXP）途径都为初级和次级类异戊二烯生物合成提供了关键代谢物。类异戊二烯代谢在膜稳定性、类固醇生物合成、维生素生产、蛋白质定位、防御和通讯、光保护、糖转运和糖蛋白生物合成中起着至关重要的作用。最近，在古细菌詹氏甲烷醛球菌中发现了MVA途径的另一个分支，涉及小分子激酶，异戊烯基磷酸激酶（IPK）。IPK属于氨基酸激酶（AAK）超家族。在体外，IPK在ATP和Mg 2+依赖性反应中磷酸化异戊烯基单磷酸（IP），产生IPP。本文描述了M. jannaschii细化到2.0−2.8 μ m的标称分辨率。值得注意的是，活性位点组氨酸残基（His 60）与底物和产物的末端磷酸盐形成氢键。该His残基用作催化磷酸或膦酸酯官能团磷酸化的AAK家族的子集的标志物;较大的家族包括羧基定向激酶，其缺乏该活性位点残基。使用H60 A、H60 N和H60 Q突变体的稳态动力学分析，His 60的Nε2氮的质子化形式被证明是催化所必需的，最有可能是通过伴随转磷酸化的过渡态的氢键稳定。此外，该结构作为IPK突变体的工程化的起点，该突变体能够从单磷酸前体化学酶促合成较长链的类异戊二烯二磷酸。
The biosynthesis of isopentenyl diphosphate (IPP) from either the mevalonate (MVA) or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway provides the key metabolite for primary and secondary isoprenoid biosynthesis. Isoprenoid metabolism plays crucial roles in membrane stability, steroid biosynthesis, vitamin production, protein localization, defense and communication, photoprotection, sugar transport, and glycoprotein biosynthesis. Recently, an alternative branch of the MVA pathway was discovered in the archaeon Methanocaldococcus jannaschii involving a small molecule kinase, isopentenyl phosphate kinase (IPK). IPK belongs to the amino acid kinase (AAK) superfamily. In vitro, IPK phosphorylates isopentenyl monophosphate (IP) in an ATP and Mg2+-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0−2.8 Å. Notably, an active site histidine residue (His60) forms a hydrogen bond with the terminal phosphate of both substrate and product. This His residue serves as a marker for a subset of the AAK family that catalyzes phosphorylation of phosphate or phosphonate functional groups; the larger family includes carboxyl-directed kinases, which lack this active site residue. Using steady-state kinetic analysis of H60A, H60N, and H60Q mutants, the protonated form of the Nε2 nitrogen of His60 was shown to be essential for catalysis, most likely through hydrogen bond stabilization of the transition state accompanying transphosphorylation. Moreover, the structures served as the starting point for the engineering of IPK mutants capable of the chemoenzymatic synthesis of longer chain isoprenoid diphosphates from monophosphate precursors.
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影响因子：
3.2
作者：
Bucurenci, N;Serina, L;Bárzu, O
通讯作者：
Bárzu, O
影响因子：
5.6
作者：
Gil-Ortiz, F;Ramón-Maiques, S;Rubio, V
通讯作者：
Rubio, V
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
影响因子：
3.2
作者：
Grochowski, LL;Xu, HM;White, RH
通讯作者：
White, RH
影响因子：
5.6
作者：
Marco-Marín, C;Gil-Ortiz, F;Rubio, V
通讯作者：
Rubio, V