De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins.

Autor: Perkins LJ; Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706., Weaver BR; Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706., Buller AR; Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706 arbuller@wisc.edu burstyn@chem.wisc.edu., Burstyn JN; Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706 arbuller@wisc.edu burstyn@chem.wisc.edu.
Jazyk: angličtina
Zdroj: Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2021 Apr 20; Vol. 118 (16).
DOI: 10.1073/pnas.2017625118
Abstrakt: 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 CoCl 2 , 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.
Competing Interests: The authors declare no competing interest.
Databáze: MEDLINE
Externí odkaz: