Structural characterization of mutations at the oxygen activation site in monomeric sarcosine oxidase .
Structural characterization of mutations at the oxygen activation site in monomeric sarcosine oxidase .
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DOI：
10.1021/bi100160j
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发表时间：
2010-05-04
期刊：
影响因子：
2.9
通讯作者：
Mathews, F. Scott
中科院分区：
文献类型：
--
作者：
Jorns, Marilyn Schuman;Chen, Zhi-wei;Mathews, F. Scott
单体肌氨酸氧化酶 (MSOX) 中的氧还原和肌氨酸氧化分别发生在黄素环 si 面和 re 面上方的不同位点。诱变研究表明 Lys265 是氧激活位点。用中性（Met、Gln、Ala）或碱性（Arg）残基取代 Lys265 会导致反应速率分别降低约 104 倍或 250 倍。用 Met 或 Arg 替换 Lys265 不会影响 MSOX 的整体结构和肌氨酸结合腔中的残基构象。 Met265 的侧链在 Lys265Met 晶体的每个分子中表现出相同的构型，并且与野生型 MSOX 中的 Lys265 几乎一致。然而，与 Lys265 相比，Arg265 的侧链发生了显着的偏移（约 4 至 5 A），指向相反的方向，并且在同一晶体的分子之间表现出显着的构象变异性。 Lys265Arg 溶液中的主要物质可能含有“翻转”的 Arg265，并且表现出可忽略不计的氧活化，类似于 Lys265Met。 Lys265Arg 观察到的氧反应性高 400 倍，归因于较小的 (< 1%) “翻转”Arg265 构象异构体，其氧反应性与野生型 MSOX 相似。在所有先前确定的 MSOX 结构中，在黄素环的 si 面上方发现了结构水 (WAT 1)，它是从 FAD:N(5) 延伸到本体溶剂的表观质子中继系统的一部分。 Lys265Met 和 Lys265Arg 中明显不存在 WAT1，这一特征可能解释了还原半反应中间体的表观动力学稳定性，该中间体可通过突变体而非野生型 MSOX 检测到。
Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-face, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 by a neutral (Met, Gln, Ala) or basic (Arg) residue results in a ~104-fold or 250-fold decrease, respectively, in reaction rate. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacing Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted (~4 to 5 A) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a “flipped-out” Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-fold higher oxygen reactivity observed with Lys265Arg is attributed to a minor (< 1%) “flipped-in” Arg265 conformer whose oxygen reactivity is similar to wild-type MSOX. A structural water (WAT 1), found above the si-face of the flavin ring in all previously determined MSOX structures, is part of an apparent proton relay system that extends from FAD:N(5) to bulk solvent. WAT1 is strikingly absent in Lys265Met and Lys265Arg, a feature that may account for the apparent kinetic stabilization of a reductive half-reaction intermediate that is detectable with the mutants but not wild-type MSOX.
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影响因子：
4.1
作者：
Dodt, G;Kim, DG;Mihalik, SJ
通讯作者：
Mihalik, SJ
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
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通讯作者：
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影响因子：
2.9
作者：
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通讯作者：
Jorns, Marilyn Schuman
影响因子：
2.9
作者：
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通讯作者：
Fagan, Rebecca L.
影响因子：
5.6
作者：
CHOTHIA, C
通讯作者：
CHOTHIA, C