Hansson A, Willows R D, Roberts T H, Hansson M
Department of Biochemistry, Lund University, P.O. Box 124, 221 00 Lund, Sweden.
Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13944-9. doi: 10.1073/pnas.212504499. Epub 2002 Sep 30.
Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins BchI, BchD, and BchH to catalyze the insertion of Mg(2+) into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that BchI forms hexamers and belongs to the ATPases associated with various cellular activities (AAA(+)) family of proteins. AAA(+) proteins are Mg(2+)-dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient BchI subunits, we demonstrate that binding of ATP is sufficient to form BchI oligomers. Further, ATPase-deficient BchI proteins can form mixed oligomers with WT BchI. The formation of BchI oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT BchI with ATPase-deficient BchI in an assay disrupts the chelatase reaction, but the presence of deficient BchI does not inhibit ATPase activity of the WT BchI. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each BchI within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient BchI are unable to perform this ATP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.
细菌叶绿素和叶绿素生物合成途径中的许多酶在整个进化过程中都得以保留，但关键步骤的分子机制仍不清楚。镁螯合酶反应就是其中一个步骤，它需要BchI、BchD和BchH蛋白在ATP水解时催化Mg(2+)插入原卟啉IX中。结构分析表明，BchI形成六聚体，属于与各种细胞活动相关的ATP酶（AAA(+)）蛋白家族。AAA(+)蛋白是Mg(2+)依赖性ATP酶，通常在ATP存在下形成寡聚环结构。通过使用ATP酶缺陷型BchI亚基，我们证明ATP的结合足以形成BchI寡聚体。此外，ATP酶缺陷型BchI蛋白可以与野生型BchI形成混合寡聚体。当与BchD和BchH结合时，BchI寡聚体的形成不足以产生镁螯合酶活性。在测定中将野生型BchI与ATP酶缺陷型BchI结合会破坏螯合酶反应，但缺陷型BchI的存在并不抑制野生型BchI的ATP酶活性。因此，六聚体每个野生型片段的ATP酶是自主的，但六聚体的所有片段都必须能够进行ATP水解才能产生镁螯合酶活性。我们认为，六聚体内每个BchI的ATP水解会导致整个六聚体发生构象变化。然而，含有ATP酶缺陷型BchI的六聚体无法进行这种依赖ATP的构象变化，镁螯合酶反应在早期就会停滞。
