| Project/Area Number |
10660082
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
応用微生物学・応用生物化学
|
|---|
| Research Institution | RIKEN (The Institute of Physical and Chemical Research) (1999)
Kyoto University (1998) |
|---|
Principal Investigator |
KATO Hiroaki Kinetic crystallography research team, Team leader (Researcher), 速度論的結晶学研究チーム, チームリーダー(研究職) (90204487)
|
|---|
| Project Period (FY) |
1998 – 1999
|
| Project Status |
Completed (Fiscal Year 1999)
|
| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
|
|---|
| Keywords | luciferase / bioluminescence / X-ray structure analysis / crystallization / 酵素反応中間体 / ATP / デヒドロルシフェリン |
|---|
| Research Abstract |
The crystal structure of Luciola cruciata firefly luciferase complexed with MgATP was determined by molecular replacement using unliganded luciferase from Photinus pyralis (sequence similarity is 67%) as a search model. The overall structure consists of two domains. Each domain is similar that of Photinus Pyralis luciferase, but a conformational change by a domain rotation with about 90 degree was caused by MgATP binding.
Firefly luciferase catalyzes the luminescence reaction through the oxidation of ィイD2DィエD2-luciferin in the presence of MgATP, and molecular oxygen. In the first step, ィイD2DィエD2-luciferin is reacted with MgATP to form an enzyme-bound luciferyl adenylate and pyrophosphate. Then the luciferyl adenylate intermediate is oxidized by molecular oxygen, and produce AMP, CoィイD2sィエD2 and an electronically excited state of oxyluciferin, which subsequently returns to the ground state with concomitant emission of visible yellow-green light. The enzyme efficiently converts chemical e
… More
nergy into light with a quantum yield of 0.88.
The luciferase from Luciola cruciata is functional as a monomer of molecular weight of about 60K with 548 amino acids. The T217I mutant enzyme was crystallized by hanging drop vapor diffusion method using PEG4000. The crystals belong to the space group P2ィイD21ィエD22ィイD21ィエD22ィイD21ィエD2 with cell constant a=58.1 AィイD4。ィエD4, b=181.7 AィイD4。ィエD4 and c=54.2 AィイD4ィエD4,. The intensity data of crystals were collected on a RIGAKU R-AXIS IIc to 2.3 AィイD4。ィエD4 resolution. The crystal structure was solved by molecular replacement using AMORE. The initial model was rebuilt by fitting correct sequence into the 2FィイD2oィエD2-FィイD2cィエD2 electron density map. The refinement of the model was carried out with X-PLOR 3.851. Current molecule includes 531 residues and the R and RィイD2freeィエD2 values are 0.23 and 0.29 respectively at 12.0-2.3 AィイD4。ィエD4 resolution. The ligand MgAMP was found in the cleft of between large and small domain. The α-phosphate oxygen are hydrogen-bonded to the side chain of His 247 and Lys517. Less
|
