气候变化领域集成服务门户(CCPortal): Archaeal Connectase is a specific and efficient protein ligase related to proteasome β subunits

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DOI	10.1073/pnas.2017871118
	Archaeal Connectase is a specific and efficient protein ligase related to proteasome β subunits
	Fuchs A.C.D.; Ammelburg M.; Martin J.; Schmitz R.A.; Hartmann M.D.; Lupas A.N.
发表日期	2021
ISSN	00278424
卷号	118 期号:11
英文摘要	Sequence-specific protein ligations are widely used to produce customized proteins "on demand." Such chimeric, immobilized, fluorophore-conjugated or segmentally labeled proteins are generated using a range of chemical, (split) intein, split domain, or enzymatic methods. Where short ligation motifs and good chemoselectivity are required, ligase enzymes are often chosen, although they have a number of disadvantages, for example poor catalytic efficiency, low substrate specificity, and side reactions. Here, we describe a sequence-specific protein ligase with more favorable characteristics. This ligase, Connectase, is a monomeric homolog of 20S proteasome subunits in methanogenic archaea. In pulldown experiments with Methanosarcina mazei cell extract, we identify a physiological substrate in methyltransferase A (MtrA), a key enzyme of archaeal methanogenesis. Using microscale thermophoresis and X-ray crystallography, we show that only a short sequence of about 20 residues derived from MtrA and containing a highly conserved KDPGA motif is required for this high-affinity interaction. Finally, in quantitative activity assays, we demonstrate that this recognition tag can be repurposed to allow the ligation of two unrelated proteins. Connectase catalyzes such ligations at substantially higher rates, with higher yields, but without detectable side reactions when compared with a reference enzyme. It thus presents an attractive tool for the development of new methods, for example in the preparation of selectively labeled proteins for NMR, the covalent and geometrically defined attachment of proteins on surfaces for cryo-electron microscopy, or the generation of multispecific antibodies. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Methanogenic archaea; Proteasome; Protein ligation; Sortase; Transpeptidase
语种	英语
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/180252
作者单位	Department of Protein Evolution, Max Planck Institute for Developmental Biology, Tubingen, 72076, Germany; Institute for General Microbiology, Christian Albrecht University of Kiel, Kiel, 24118, Germany
推荐引用方式 GB/T 7714	Fuchs A.C.D.,Ammelburg M.,Martin J.,et al. Archaeal Connectase is a specific and efficient protein ligase related to proteasome β subunits[J],2021,118(11).
APA	Fuchs A.C.D.,Ammelburg M.,Martin J.,Schmitz R.A.,Hartmann M.D.,&Lupas A.N..(2021).Archaeal Connectase is a specific and efficient protein ligase related to proteasome β subunits.Proceedings of the National Academy of Sciences of the United States of America,118(11).
MLA	Fuchs A.C.D.,et al."Archaeal Connectase is a specific and efficient protein ligase related to proteasome β subunits".Proceedings of the National Academy of Sciences of the United States of America 118.11(2021).