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.
CRP and FNR are well-characterized members of the CRP/FNR family of regulatory proteins that function to maximize cellular energy production. In this study, we identify several new subgroups of the CRP/FNR family, many of which have not yet been characterized.
The CRP/FNR family of regulatory proteins constitutes a large collection of related transcription factors, several of which globally regulate cellular energy production. A well-characterized example is FNR (called FnrL in
Rhodobacter capsulatus
), which is responsible for regulating the expression of numerous genes that promote maximal energy production and growth under anaerobic conditions.
RcoM,
a heme-containing, CO-sensing transcription factor, is one
of two known bacterial regulators of CO metabolism. Unlike its analogue
CooA, the structure and DNA-binding properties of RcoM remain largely
uncharacterized. Using a combination of size exclusion chromatography
and sedimentation equilibrium, we demonstrate that RcoM-1 from Paraburkholderia xenovorans is a dimer, wherein the
heme-binding domain mediates dimerization. Using bioinformatics, we
show that RcoM is found in three distinct genomic contexts, in accordance
with the previous literature. We propose a refined consensus DNA-binding
sequence for RcoM based on sequence alignments of coxM-associated promoters. The RcoM promoter consensus sequence bears
two well-conserved direct repeats, consistent with other LytTR domain-containing
transcription factors. In addition, there is a third, moderately conserved
direct repeat site. Surprisingly, PxRcoM-1 requires
all three repeat sites to cooperatively bind DNA with a [P]1/2 of 250 ± 10 nM and an average Hill coefficient, n, of 1.7 ± 0.1. The paralog PxRcoM-2
binds to the same triplet motif with comparable affinity and cooperativity.
Considering this unusual DNA binding stoichiometry, that is, a dimeric
protein with a triplet DNA repeat-binding site, we hypothesize that
RcoM interacts with DNA in a manner distinct from other LytTR domain-containing
transcription factors.
Carbon monoxide (CO) serves as a source of energy and carbon for a diverse set of microbes found in anaerobic and aerobic environments. The enzymes that bacteria and archaea use to oxidize CO depend upon complex metallocofactors that require accessory proteins for assembly and proper function.
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