De novo biosynthesis of a nonnatural cobalt porphyrin cofactor inE. coliand incorporation into hemoproteins

Abstract

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.

Photocatalytic Hydrogen Production and Carbon Dioxide Reduction Catalyzed by an Artificial Cobalt Hemoprotein

“…As shown in Table 1 , the electronic absorption spectrum of the oxidized Co(III)–(A3A3′)Y26C- 4 biohybrid was similar to those obtained through the incorporation of Co(III)PPIX not only into apoproteins, which bind the cobalt cation through an axial thiolate ligand such as P450 cam [ 26 ] and CYP 119 [ 27 ], but also to those of some apoproteins that bind the cobalt cation through an axial histidine ligand such as Mb, HRP [ 24 ], and DYP [ 28 ]. Such a comparison does not allow for the identification the cobalt axial ligand of cobalt in (A3A3′)Y26C- 4 .…”

“…The characteristics of these spectra are compared in Table 1 with those obtained after the incorporation of Co(III)– and Co(II)–protoporphyrin IX into the apoprotein of various hemoproteins including myoglobin (Mb) [ 24 ] and its H64V-V68A double mutant [ 25 ]; two cytochromes P450, P450 Cam [ 26 ] and CYP119 [ 27 ]; two peroxidases, horseradish peroxidase (HRP) [ 24 ] and dye decolorizing peroxidase (DYP) [ 28 ], aldoxime dehydrogenase (Oxd) [ 28 ] and catalase (Cat) [ 28 ].…”

Heme-Based Gas Sensors in Nature and Their Chemical and Biotechnological Applications

“…One strategy to explore this is to replace the native heme by another metalloporphyrin analogs, providing novel functions to the protein as described before. To achieve this, engineered E. coli strains or growth conditions have been explored to facilitate direct incorporation of heme analogs or production of hemeless proteins [139][140][141][142]. The engineered RP523 strain of E. coli was prepared, exhibiting disruption of biosynthesis of the heme (hemB and hemC) along with the heme permeability phenotype [139].…”

Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues

“…The BL21­(DE3) strain of E. coli . can biosynthesize Co­(PPIX) and subsequently incorporate it into a variety of heme proteins . In contrast to these methods, the RP523 expression system does not rely on bacterial systems to acquire or synthesize the cofactor, enabling precise tuning of heme proteins for a variety of applications.…”