T. Tsukihara, et al., Science 269, 1069-1074 (1995) S. Iwata et. al., Nature 376, 660-669 (1995)
▪ Bovine heart CcO (PDB ID:1OCC): membrane protein ▪ 13 subunits, MW = 200 kDa (1850 amino acids)
Why designing artificial biocatalysts?
➢ Approaches to novel biocatalysts: Top down: reprogramming native enzymes Bottom up: design and engineering artificial biocatalysts
化学生物学与合成生物学 王江云
人工合成生命的重大科学意义
近40亿年的进化历程，创造了丰 富多彩的生物。据估计地球物种 总数多达1亿种。
Age of Age of mammals Insects andreptiles
amphibians invade the land
Plants invade the land
人工合成生命的重大科学意义
• 改造生命，造福人类 • 快速发展的领域，生命科学的新方法 • 工程、生物和信息科学的多学科综合交叉 • 产生广泛的工业应用
化工
新能源
人
工
生
命
健康 环境
新材料
绿色农业
新医药
我们应该做什么？
• 能量利用水平是人类进步的标志 • 电能不能在自然界稳定存在，自然进化生物不能高效利用电能 • 人工合成电能、光能、生物质能高效利用的新生命体系具有重
large-scale industrial applications
Bakers’ yeast vs. white rot fungi
Bakers’ yeast
White rot fungi
Bakers’ yeast is much cheaper and widely available for industrial applications
➢ Structural features (bond distance, angle and geometry) vary widely and ill-defined;
➢ Most metal ions have beautiful colors and strong magnetic properties, serving as in situ probe of the design process.
Cytochrome c peroxidase (CcP) Manganese peroxidase (MnP)
CcP (baker’s yeast)
Oxidation of cyt c
MnP (white rot fungi)
Degradation of lignin and many aromatic pollutants including PCBs
Overcoming over-potential problem: rationally tuning redox potentials without affecting the active site
Potential (mV) Eo (mV vs. NHE)
N47S F114N M121L M121X
H3CO
OH
O HO
OH H3CO
OH
H3CO
O
OH H3CO O
OCH3
O
O
HO
OCH3 O
OH
HO O
HO O
HO O O
O
O O
HO H2 O
O O
O
OH
HO OH
OH
OH
O H3CO
HO OCH3
OCH3 O
HO
O
O
OH
OH
H3CO
O O
O O
O
O O
O
OCH3
OH
HO OCH3
OH
O
OH
HO OH
700
600
+ ~140 mV
600
N47S M121X
500
+ ~140 mV
400
400
M121Q
M121X
~110 mV
300
Native azurin
F114P M121X
200
200
100
0
-1
0
1
2
3
Log P
NicholYa.sLMu,. eMtaarls. hNaallt,uDreew46a0in, 8K5. 5G-8a6rn2e(r2, 0T0iff9a)n. y D. Wilson, Yi-Gui Gao, Howard Robinson, Mark J. Nilges, Yi Lu Nature 462, 113-116 (2009).
(native CuA, 2.7 Å)
(designed CuA, 1.65 Å)
The designed CuA looks almost identical to the native CuA.
Iwata, S.; Ostermeier, C.; Ludwig, B.; Michel, H. Nature 376, 660-669 (1995). Y.HLauy,,eMt a. Tl..N; Raitcuhrear4d6, 0J,. 8H5.;5a-8n6d2L(u2,0Y0.9)P. roc. Natl. Acad. Sci. U. S. A. 93, 461-464 (1996).
要意义。
电能
风能
化能自养
太阳能
光能自养 有氧代谢
新人工生命体系
水能 核能
二氧化碳还原酶在电极上的定点偶联
利用电能进行淀粉生物合成
形成电能细胞，创新生物的能量来源，引领下一代生物技 术的发展，如生物计算机、生物传感器、分子马达等。
电子催化酶与电子传递通道设计
电能驱动
线粒体基因组人工合成与优化组装
C. H. Kjaergaard, J. Rossmeisl, and J. K. Nørskov, Inorg. Chem. 49, 3567–3572 (2010).
Cytochrome c Oxidase (CcO)
CuA center
His240
His291
His290
CuB Tyr244
Y. Lu, et al. Nature 460, 855-862 (2009).
Challenges and opportunities in designing metalloenzymes as artificial biocatalysts
➢ A wide number of metal ions and difference oxidation states of the same metal ions;
Fossils present but rare
Evolution and expansion of life
亚里斯多德
达尔文 沃森克里克
人类对生命本质 的研究有两千多 年了。基因组测 序之后，我们对 生物的认识更加 深入，而对生命 本质的理解还远 远不够
人类基因组 文特
千人基因组
不能创造的东西就不能被理解。 人工生命可以更好理解生命本质。
HO
H3CO
O
O
OH
HO
OCH3
O O
OCH3
Lignin is the second most abundant biopolymer on earth next to cellulose
A critical barrier to biomass conversion
While rot fungi is known degrade lignin naturally, but is very difficult for
Xiaohong Liu, Yang Yu, Cheng Hu, Wei Zhang, Yi Lu, Jiangyun Wang Angew. Chem., Int. Ed. 51, 4312-4316 (2012).
Lignin biodegradation
HO
O
HO
O
OH
HO O
HO
O
OБайду номын сангаас
White rot fungi
How good is it?
Sohini Mukherjee, Sabyasachi Bandyopadhyay, Arnab Mukherjee, Yi Lu, Abhishek D
How good is it?
Catalyst Native CcO Synthetic model Designed protein
1. B. C. Finzel, T. L. Poulos, and J. Kraut, J. Biol. Chem. 259, 13027 (1984). 2. M. Sundaramoorthy, K. Kishi, M. H. Gold, and T. L. Poulos, J. Biol. Chem.269, 32759 (1994).
(cytochrome c oxidase hydrogenase)
(photosystem II)
Most important enzymes for sustainable energy are metalloenzymes. However, metalloenzymes are too expensive.