Kinetic analysis of a globin-coupled diguanylate cyclase, YddV: Effects of heme iron redox state, axial ligands, and heme distal mutations on catalysis
| Autor: | Jakub Vavra, Marketa Martinkova, Toru Shimizu, Václav Martínek, Veronika Fojtikova-Proskova, Alzbeta Lengalova, Martin Stranava |
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| Rok vydání: | 2019 |
| Předmět: |
Heme binding
Diguanylate cyclase activity Stereochemistry Iron Heme 010402 general chemistry Ligands 01 natural sciences Biochemistry Redox Inorganic Chemistry chemistry.chemical_compound Maltose-binding protein Catalytic Domain Enzyme kinetics Globin biology 010405 organic chemistry Escherichia coli Proteins 0104 chemical sciences Kinetics chemistry Amino Acid Substitution biology.protein Diguanylate cyclase Phosphorus-Oxygen Lyases Oxidation-Reduction Protein Binding |
| Zdroj: | Journal of inorganic biochemistry. 201 |
| ISSN: | 1873-3344 |
| Popis: | 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(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 YddV-MBP 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. |
| Databáze: | OpenAIRE |
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