Heme pocket hydrogen bonding residue interactions within the Pectobacterium Diguanylate cyclase-containing globin...

Area of Science:

• Biochemistry
• Microbiology
• Molecular Biology

Background:

• Heme-based sensor proteins regulate cellular responses to gaseous environments.
• Globin-coupled sensors (GCS) are bacterial oxygen sensors composed of a globin domain linked to output domains.
• Diguanylate cyclase domains synthesize cyclic-di-GMP (c-di-GMP), a second messenger regulating bacterial biofilm formation.

Purpose of the Study:

• To investigate the role of heme pocket residues in modulating the activity of the diguanylate cyclase domain in Pectobacterium carotovorum GCS (PccGCS).
• To elucidate the relationship between heme pocket conformation, flexibility, and GCS signaling.
• To identify key residues involved in ligand-dependent GCS signaling.

Main Methods:

• Enzyme kinetics were used to characterize PccGCS variants.
• Resonance Raman (rR) spectroscopy was employed to analyze heme pocket properties.
• Site-directed mutagenesis was performed to create variants of PccGCS.

Main Results:

• Specific heme pocket residues, including those involved in hydrogen bonding and heme edge interactions, were identified as critical modulators of PccGCS activity.
• Changes in heme pocket conformation and flexibility were correlated with altered GCS activity.
• Residue substitutions affected the oxygen-sensing capabilities of the protein.

Conclusions:

• Heme pocket residues play a crucial role in controlling the activity of the diguanylate cyclase domain in GCS proteins.
• Understanding these residue-function relationships provides insights into the oxygen-sensing mechanism of GCS.
• This knowledge may facilitate the development of strategies to control O2-dependent c-di-GMP production and biofilm formation.