title: Heme interacts with histidine- and tyrosine-based protein motifs and inhibits enzymatic activity of chloramphenicol acetyltransferase from Escherichia coli.
abstract: BACKGROUND
  The occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements.
  
  
  METHODS
  To understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV-vis, resonance Raman, cw-EPR and 2D NMR spectroscopy.
  
  
  RESULTS
  We observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37 μM.
  
  
  CONCLUSIONS
  It was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme.
  
  
  CONCLUSIONS
  Understanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.
authors.affiliation: Pharmaceutical Chemistry I, Institute of Pharmacy, University of Bonn, 53119 Bonn, Germany.
authors.forename: Hans Henning
authors.initials: HH
authors.lastname: Brewitz
authors.affiliation: Leibniz Institute on Aging, Fritz Lipmann Institute, 07745 Jena, Germany.
authors.forename: Nishit
authors.initials: N
authors.lastname: Goradia
authors.affiliation: Institute of Physical and Theoretical Chemistry, University of Bonn, 53115 Bonn, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Schubert
authors.affiliation: Center for Sepsis Control and Care (CSCC), Jena University Hospital, 07747 Jena, Germany; Leibniz Institute of Photonic Technology, 07745 Jena, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Galler
authors.affiliation: Centre National de la Recherche Scientifique (CNRS), Bioénergetique et Ingenierie des Protéines, UMR 7281, 13009 Marseille, France.
authors.forename: Toni
authors.initials: T
authors.lastname: Kühl
authors.affiliation: Pharmaceutical Chemistry I, Institute of Pharmacy, University of Bonn, 53119 Bonn, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Syllwasschy
authors.affiliation: Center for Sepsis Control and Care (CSCC), Jena University Hospital, 07747 Jena, Germany; Leibniz Institute of Photonic Technology, 07745 Jena, Germany; Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University Jena, 07743 Jena, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Popp
authors.affiliation: Center for Sepsis Control and Care (CSCC), Jena University Hospital, 07747 Jena, Germany; Leibniz Institute of Photonic Technology, 07745 Jena, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Neugebauer
authors.affiliation: Institute of Physical and Theoretical Chemistry, University of Bonn, 53115 Bonn, Germany.
authors.forename: Gregor
authors.initials: G
authors.lastname: Hagelueken
authors.affiliation: Institute of Physical and Theoretical Chemistry, University of Bonn, 53115 Bonn, Germany.
authors.forename: Olav
authors.initials: O
authors.lastname: Schiemann
authors.affiliation: Leibniz Institute on Aging, Fritz Lipmann Institute, 07745 Jena, Germany. Electronic address: oliver.ohlenschlaeger@leibniz-fli.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Ohlenschläger
authors.affiliation: Pharmaceutical Chemistry I, Institute of Pharmacy, University of Bonn, 53119 Bonn, Germany. Electronic address: dimhof@uni-bonn.de.
authors.forename: Diana
authors.initials: D
authors.lastname: Imhof
mesh_terms: D020816:Amino Acid Motifs; D015500:Chloramphenicol O-Acetyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D006418:Heme / Q000494:pharmacology*; D006639:Histidine; D009682:Magnetic Resonance Spectroscopy; D013059:Spectrum Analysis, Raman; D014443:Tyrosine
chemical_list: D004791:Enzyme Inhibitors; D014443:Tyrosine; D006418:Heme; D006639:Histidine; D015500:Chloramphenicol O-Acetyltransferase
keywords: Chloramphenicol acetyltransferase; Heme-peptide/protein complex; Heme-regulatory motif (HRM); Histidine-based HRM; Tyrosine-based HRM