The FG loop of a heme-based gas sensor enzyme, Ec DOS, functions in heme binding, autoxidation and catalysis.
Ec DOS is a heme-based gas sensor enzyme that catalyzes conversion from cyclic-di-GMP to linear-di-GMP in response to gas molecules, such as oxygen, CO and NO. Ec DOS contains an N-terminal heme-binding PAS domain and C-terminal phosphodiesterase domain. Based on crystal structures of the isolated heme-binding domain, it is suggested that the FG loop is involved in intra-molecular signal transduction to the catalytic domain. We generated nine full-length proteins mutated at ionic and non-ionic polar residues between positions 83 and 96 corresponding to the F-helix and FG loop, and examined the heme binding properties, autoxidation rates, and catalytic activities of mutant proteins. N84A and R85A mutant proteins displayed lower heme binding affinities, consistent with the finding that Asn84 interacts with propionate of protoporphyrin IX, and Arg85 with Asp40 on the heme proximal side. Autoxidation rates (0.058-0.54 min(-1)) of R91A, S96A and K89A/R91A/E93A mutant proteins were significantly higher than that (0.0053 min(-1)) of wild-type protein, suggesting that these residues in the FG loop form heme distal architecture conferring stability to the Fe(II)-O(2) complex. Catalytic activities of N84A and R85A mutant proteins with low heme affinity were significantly higher than those of wild-type protein in the absence of gas molecules. Accordingly, we propose that loss of heme binding enhances basal catalysis without the gas molecule, consistent with previous reports on heme inhibition of Ec DOS catalysis.
Ito
Shinya
S
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Sendai 980-8577, Japan.
Igarashi
Jotaro
J
Shimizu
Toru
T
D019943:Amino Acid Substitution; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics
D029968:Escherichia coli Proteins; D006418:Heme; D002248:Carbon Monoxide; D010727:Phosphoric Diester Hydrolases; C464427:dosP protein, E coli; D010100:Oxygen