Open AccessControl of Metabolite Flux during the Final Steps of Heme b Biosynthesis in Gram-Positive Bacteria
Author(s) -
Arianna I. Celis,
Jacob E. Choby,
James Kentro,
Eric P. Skaar,
Jennifer L. DuBois
Publication year - 2019
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.9b00140
Subject(s) - heme , biochemistry , enzyme , ferrochelatase , chemistry , mutant , bacteria , metabolite , biosynthesis , biology , microbiology and biotechnology , gene , genetics
The pathway for assembling heme ends with a unique set of enzymes in Gram-positive bacteria. Substrates for these reactions include coproporphyrin III, Fe(II), and H 2 O 2 , which are highly reactive and toxic. Because these bacteria lack membranous compartments, we hypothesized that metabolite flux may occur via a transient protein-protein interaction between the final two pathway enzymes, coproporphyrin ferrochelatase (CpfC) and coproheme decarboxylase (ChdC). This hypothesis was tested using enzymes from the pathogen Staphylococcus aureus and a corresponding Δ chdC knockout strain. The ultraviolet-visible spectral features of coproporphyrin III served as an in vitro indicator of a protein-protein interaction. A CpfC-ChdC K D of 17 ± 7 μM was determined, consistent with transient complexation and supported by the observation that the catalytic competence of both enzymes was moderately suppressed in the stable complex. The Δ chdC S. aureus was transformed with plasmids containing single-amino acid mutants in the active site gate of ChdC. The porphyrin content and growth phenotypes of these mutants showed that K129 and Y133 promote the ChdC-CpfC interaction and revealed the importance of E120. Understanding the nature of interactions between these enzymes and those further upstream in the heme biosynthesis pathway could provide new means of specifically targeting pathogenic Gram-positive bacteria such as S. aureus .