气候变化领域集成服务门户(CCPortal): De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

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DOI	10.1073/pnas.2017625118
	De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins
	Perkins L.J.; Weaver B.R.; Buller A.R.; Burstyn J.N.
发表日期	2021
ISSN	00278424
卷号	118 期号:16
英文摘要	Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Artificial metalloenzymes; Cobalt porphyrin; Heme; Protein expression
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/179833
作者单位	Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, United States
推荐引用方式 GB/T 7714	Perkins L.J.,Weaver B.R.,Buller A.R.,et al. De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins[J],2021,118(16).
APA	Perkins L.J.,Weaver B.R.,Buller A.R.,&Burstyn J.N..(2021).De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins.Proceedings of the National Academy of Sciences of the United States of America,118(16).
MLA	Perkins L.J.,et al."De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins".Proceedings of the National Academy of Sciences of the United States of America 118.16(2021).