Kinetic analysis of a globin-coupled diguanylate cyclase, YddV: Effects of heme iron redox state, axial ligands, and heme distal mutations on catalysis.

Kinetic analysis of a globin-coupled diguanylate cyclase, YddV: Effects of heme iron redox state, axial ligands, and heme distal mutations on catalysis. J Inorg Biochem. 2019 Sep 06;201:110833 Authors: Lengalova A, Fojtikova-Proskova V, Vavra J, Martínek V, Stranava M, Shimizu T, Martinkova M Abstract Heme-based oxygen sensors allow bacteria to regulate their activity based on local oxygen levels. YddV, a globin-coupled 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 KmGTP 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(II...
Source: Journal of Inorganic Biochemistry - Category: Biochemistry Authors: Tags: J Inorg Biochem Source Type: research