Designer installation of a substrate recruitment domain to tailor enzyme specificity

被引:10
作者
Park, Rodney [1 ]
Ongpipattanakul, Chayanid [2 ,7 ]
Nair, Satish K. [2 ,3 ,4 ]
Bowers, Albert A. [5 ]
Kuhlman, Brian [1 ,6 ]
机构
[1] Univ N Carolina, Dept Biochem & Biophys, Sch Med, Chapel Hill, NC 27599 USA
[2] Univ Illinois, Dept Biochem, Urbana, IL USA
[3] Univ Illinois, Ctr Biophys & Computat Biol, Urbana, IL USA
[4] Univ Illinois, Inst Genom Biol, Urbana, IL USA
[5] Univ North Carolina Chapel Hill, UNC Eshelman Sch Pharm, Div Chem Biol & Med Chem, Chapel Hill, NC 27599 USA
[6] Univ North Carolina Chapel Hill, Lineberger Comprehens Canc Ctr, Chapel Hill, NC 27599 USA
[7] Univ Calif San Francisco, Sch Pharm, San Francisco, CA USA
基金
美国国家科学基金会;
关键词
CATECHOL O-METHYLTRANSFERASE; SH3; DOMAIN; BINDING; MODEL; PHOSPHORYLATION; PEPTIDES; SOFTWARE; KINETICS; INSIGHTS; PATHWAY;
D O I
10.1038/s41589-022-01206-0
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Promiscuous enzymes that modify peptides and proteins are powerful tools for labeling biomolecules; however, directing these modifications to desired substrates can be challenging. Here, we use computational interface design to install a substrate recognition domain adjacent to the active site of a promiscuous enzyme, catechol O-methyltransferase. This design approach effectively decouples substrate recognition from the site of catalysis and promotes modification of peptides recognized by the recruitment domain. We determined the crystal structure of this novel multidomain enzyme, SH3-588, which shows that it closely matches our design. SH3-588 methylates directed peptides with catalytic efficiencies exceeding the wild-type enzyme by over 1,000-fold, whereas peptides lacking the directing recognition sequence do not display enhanced efficiencies. In competition experiments, the designer enzyme preferentially modifies directed substrates over undirected substrates, suggesting that we can use designed recruitment domains to direct post-translational modifications to specific sequence motifs on target proteins in complex multisubstrate environments.
引用
收藏
页码:460 / +
页数:16
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