National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01GM138990
米国
Department of Energy (DOE, United States)
DE-AC02-76SF00515
米国
Department of Energy (DOE, United States)
DE-AC02-05CH11231
米国
Department of Energy (DOE, United States)
DE-AC02-06CH11357
米国
引用
ジャーナル: Science / 年: 2021 タイトル: Modular polyketide synthase contains two reaction chambers that operate asynchronously. 著者: Saket R Bagde / Irimpan I Mathews / J Christopher Fromme / Chu-Young Kim / 要旨: Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and β-keto ...Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and β-keto group modification reactions. We determined the 2.4-angstrom-resolution x-ray crystal structure and the 3.1-angstrom-resolution cryo–electron microscopy structure of the Lsd14 polyketide synthase, stalled at the transacylation and condensation steps, respectively. These structures revealed how the constituent domains are positioned relative to each other, how they rearrange depending on the step in the reaction cycle, and the specific interactions formed between the domains. Like the evolutionarily related mammalian fatty acid synthase, Lsd14 contains two reaction chambers, but only one chamber in Lsd14 has the full complement of catalytic domains, indicating that only one chamber produces the polyketide product at any given time.
The authors state that there are two copies of the multidomain modular polyketide synthase Lsd14 in ...The authors state that there are two copies of the multidomain modular polyketide synthase Lsd14 in the unit cell, forming a dimer. The Lsd14 polypeptide chain is 1667 residues in length and includes the ketosynthase (KS), acyltransferase (AT), ketoreductase (KR), acyl carrier protein (ACP) domains, and the linkers connecting these domains. We modelled two copies each of KS, AT and KR and one copy of the ACP. We were not able to model the second ACP and parts of AT-to-KR and KR-to-ACP linkers due to lack of clear electron density map. Hence we were not able to assign polypeptide chain identity for the entire sequence. We built the KS-AT di-domains, KR and the ACP with separate chain IDs.
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実験情報
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実験
実験
手法: X線回折 / 使用した結晶の数: 1
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試料調製
結晶
マシュー密度: 2.19 Å3/Da / 溶媒含有率: 43.9 %
結晶化
温度: 291 K / 手法: 蒸気拡散法, シッティングドロップ法 / pH: 4 詳細: 0.2 M lithium sulfate, 0.015 M magnesium sulfate, 0.1 M sodium acetate, pH 4.0, and 22% polyacrylic acid 5100
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データ収集
回折
ID
平均測定温度 (K)
Crystal-ID
Serial crystal experiment
1
100
1
N
2
100
1
N
3
100
1
N
放射光源
由来
サイト
ビームライン
ID
波長 (Å)
シンクロトロン
SSRL
BL9-2
1
0.9795
シンクロトロン
ALS
5.0.2
2
1.005
シンクロトロン
APS
17-ID
3
1
検出器
タイプ
ID
検出器
日付
DECTRIS PILATUS 6M
1
PIXEL
2018年5月2日
DECTRIS PILATUS3 6M
2
PIXEL
2018年3月15日
DECTRIS PILATUS 6M
3
PIXEL
2018年2月17日
放射
ID
プロトコル
単色(M)・ラウエ(L)
散乱光タイプ
Wavelength-ID
1
SINGLEWAVELENGTH
M
x-ray
1
2
SINGLEWAVELENGTH
M
x-ray
2
3
SINGLEWAVELENGTH
M
x-ray
3
放射波長
ID
波長 (Å)
相対比
1
0.9795
1
2
1.005
1
3
1
1
反射
解像度: 2.35→39.2 Å / Num. obs: 130003 / % possible obs: 98.1 % / 冗長度: 26.3 % / CC1/2: 0.999 / Net I/σ(I): 18.5
反射 シェル
解像度: 2.35→2.41 Å / 冗長度: 15.2 % / Mean I/σ(I) obs: 1.3 / Num. unique obs: 9300 / CC1/2: 0.683 / % possible all: 94.2
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解析
ソフトウェア
名称
バージョン
分類
BUSTER
2.10.4
精密化
XDS
データ削減
autoPROC
データ削減
XDS
データスケーリング
autoPROC
データスケーリング
PHENIX
位相決定
Coot
モデル構築
精密化
構造決定の手法: 単波長異常分散 / 解像度: 2.35→39.2 Å / Cor.coef. Fo:Fc: 0.945 / Cor.coef. Fo:Fc free: 0.929 / 交差検証法: THROUGHOUT / SU R Blow DPI: 0.334 / SU Rfree Blow DPI: 0.227