Kinetic analysis of a globin-coupled diguanylate cyclase, YddV: Effects of heme iron redox state, axial ligands, and heme distal mutations on catalysis
Abstract
Heme-based oxygen sensors allow bacteria to regulate their activity based on local oxygen levels. YddV, a globincoupled oxygen sensor with diguanylate cyclase activity from Escherichia coli, regulates cyclic-di-GMP synthesis based on oxygen availability.
Stable and active samples of the full-length YddV protein were prepared by attaching it to maltose binding protein (MBP). To better understand the full-length protein's structure, the interactions between its domains were examined by performing a kinetic analysis.
The diguanylate cyclase reaction catalyzed by YddV MBP exhibited Michaelis-Menten kinetics. Its pH optimum was 8.5-9.0, and catalysis required either Mg2+ or Mn2+; other divalent metal ions gave no activity.
The most active form of YddV-MBP had a 5-coordinate Fe(III) heme complex; its kinetic parameters were Km GTP 84 +- 21 μM and kcat 1.2 min-1. YddV-MBP with heme Fe(II), heme Fe(II)-O2, and heme Fe(II)-CO complexes had kcat values of 0.3 min-1, 0.95 min-1, and 0.3 min-1, respectively, suggesting that catalysis is regulated by the heme iron's redox state and axial ligand binding.
The kcat values for heme Fe(III) complexes of L65G, L65Q, and Y43A YddV-MBP mutants bearing heme distal amino acid replacements were 0.15 min-1, 0.26 min-1 and 0.54 min-1, respectively, implying that heme distal residues play key regulatory roles by mediating signal transduction between the sensing and functional domains. Ultracentrifugation and size exclusion chromatography experiments showed that YddVMBP is primarily dimeric in solution, with a sedimentation coefficient around 8.
The inactive heme-free H93A mutant is primarily octameric, suggesting that catalytically active dimer formation requires heme binding.