Abstract
The biogenesis of molybdenum-containing enzymes is a sophisticated process involving the insertion of a complex molybdenum cofactor into competent apoproteins. As for many molybdoenzymes, the maturation of trimethylamine-oxide reductase TorA requires a private chaperone. This chaperone (TorD) interacts with the signal peptide and the core of apo-TorA. Using random mutagenesis, we established that alpha-helix 5 of TorD plays a key role in the core binding and that this binding drives the maturation of TorA. In addition, we showed for the first time that TorD interacts with molybdenum cofactor biosynthesis components, including MobA, the last enzyme of cofactor synthesis, and Mo-molybdopterin, the precursor form of the cofactor. Finally we demonstrated that TorD also binds the mature molybdopterin-guanine dinucleotide form of the cofactor. We thus propose that TorD acts as a platform connecting the last step of the synthesis of the molybdenum cofactor just before its insertion into the catalytic site of TorA.
Highlights
TorA is a bis-MGD-containing reductase involved in periplasmic bacterial trimethylamine oxide (TMAO) respiration, and TorD is the dedicated chaperone allowing optimal production of active TorA in the cell (13, 20)
TorDϪ strain (LCB515) cannot use TMAO as a substrate when grown at 43 °C unless it bears a plasmid carrying and expressing torD gene (18)
Because GTP and Mo-MPT bind TorD and because MobA catalyzed the attachment of GMP to the Mo-MPT from GTP, we investigated whether TorD interacts with E. coli MobA. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride biochemical cross-linking with purified TorD and MobA revealed that the two proteins interacted (Fig. 7)
Summary
E. coli TorD protein is part of a large family of metalloprotein-associated chaperones presenting a low homology level in their primary amino acid sequences but sharing an all ␣-helical structure organized in two domains connected by a short hinge region (23–25). Site-directed mutagenesis showed that this interdomain region with two conserved residues was critical for the signal sequence recognition and the proofreading activity of TorD (21). A poorly conserved patch in TorD protein was shown to interact with the core of apoTorA but not with the signal peptide. By using several in vitro and in vivo approaches, we revealed that the maturation of the TorA apoprotein is tightly dependent on this interaction. It appears that TorD has a third function. It is a Moco-binding protein capable of specific interaction with MobA, the enzyme involved in MGD biosynthesis. Our work reveals a new role for members of the TorD family
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