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.

General significance: 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.

Keywords: Chloramphenicol acetyltransferase; Heme-peptide/protein complex; Heme-regulatory motif (HRM); Histidine-based HRM; Tyrosine-based HRM.