Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 118, Issue 16, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2017625118
Keywords
artificial metalloenzymes; cobalt porphyrin; protein expression; heme
Categories
Funding
- Office of the Vice Chancellor for Research and Graduate Education at the University of Wisconsin-Madison
- Wisconsin Alumni Research Foundation
- NSF [DGE-1747503]
- Shimadzu's Academic Grant Program
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This study introduces a novel method for biosynthesizing cobalt protoporphyrin IX (CoPPIX) using Escherichia coli BL21(DE3) under specific growth conditions. The newly synthesized CoPPIX has been successfully incorporated into multiple proteins, providing a new pathway for the synthesis of nonnatural cofactors.
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.
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