The modification and targeting of complex metal-cofactors into their apo-enzymes
Final Report Abstract
Molybdoenzymes are widespread in all domains of life and catalyze key steps in carbon, sulfur and nitrogen metabolism. In all molybdoenzymes binding the molybdenum cofactor (Moco), their active site possesses a molybdenum atom coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). According to the different modifications in the ligand sphere of the molybdenum atom or at the pterin backbone, molybdoenzymes have been classified into three families: the xanthine oxidase (XO) family, the sulfite oxidase (SO) family and the dimethyl sulfoxide (DMSO) reductase family. In Escherichia coli 19 molybdoenzymes are present that group to all three families, with the majority of enzymes categorizing to the DMSO reductase family. In the DMSO reductase family, almost all enzymes bind a guanine nucleotide derivative of Mo-MPT named the MPT guanine dinucleotide (MGD) cofactor. The DMSO reductase family is diverse in both structure and function, but all members have two equivalents of the pterin cofactor bound to the metal, forming the so-called bis-MGD or bis-Mo-MPT cofactor. The molybdenum coordination sphere is usually completed by a single Mo=O group with a sixth ligand in the MPT2-MoVIO(X) core, however, a sulfido-ligand has been recently described to replace the oxo-group in several molybdoenzymes in E. coli. The sixth ligand, X, is predominantly an amino acid side chain from the protein backbone, like a serine, a cysteine, a selenocysteine, an aspartate but a hydroxide and/or water molecule has also been identified. The reactions catalyzed by members of this family mainly involve oxygen-atom transfer reactions, but C-H bond cleavage reactions also occur. Members of the DMSO reductase family in E. coli include, e.g. the dissimilatory nitrate reductases (NarGHI, NarZYV, NapAB), formate dehydrogenases (FdhF, FdnGHI, FdoGHI), trimethylamine-N-oxide (TMAO) reductase (TorA), DMSO reductase (DmsABC), selenate reductase (YnfEF) and the biotin sulfoxide reductase (BisC). A novel modification of enzymes of the DMSO reductase family that has been identified by us during the funding period. We were able to reveal a modification of Moco by a sulfur modification of bis-MGD in more enzymes of the DMSO reductase family. We identified this modification to be present in E. coli TMAO reductase. The role of the sulfur ligand for the catalytic mechanism of these enzymes is also not completely understood. For formate dehydrogenases, a role as hydride- or proton acceptor after C-H bond cleavage of the substrate has been suggested. For TMAO reductase, we proposed that the sulfido-ligand triggers the redoxpotential of the molybdenum atom. So far, it is not completely understood how the sulfido-ligand is inserted into bis-MGD of TMAO reductase. Overall, we suggest that the sulfido-ligand at the bis-MGD cofactor is more common to this group of enzymes than previously expected. For the insertion of Moco into target enzymes, it was proposed that this step is catalyzed by Moco-binding molecular chaperones, which bind the respective Moco variant and insert it into the target molybdoenzyme. With a few exceptions, most of the molybdoenzymes of the DMSO reductase family in E. coli have a specific bis-MGD binding chaperone for Moco insertion, which in some cases is shared between several molybdoenzymes: NarJ is the chaperone for nitrate reductase A composed of NarGHI; NarW is the chaperone for nitrate reductase Z, composed of NarZYV; DmsD is the chaperone for DmsABC and YnfEF; FdhD is the chaperone for FdhF, FdoGHI and FdnGH, and TorD the chaperone for TorA. However, exceptions occur since no chaperone has been identified for the cytosolic biotin sulfoxide reductase BisC so far. It has been suggested that these chaperones not only facilitate the insertion of bis-MGD into the target enzyme, but also directly bind bis-MGD. The insertion of the terminal sulfido-ligand to the molybdenum atom likely occurs while the cofactor is bound to the chaperone. For the E. coli FdhD protein it was reported that it acts as a sulfur transferase between the L-cysteine desulfurase IscS and the target enzyme FdhF. We were able to show during the funding period, that the sulfido-containing form of bis-MGD is bound to the homologous chaperones FdsC from Rhodobacter capsulatus and FdhD from E. coli. We suggested that bis-MGD binding to these chaperones for enzymes of the DMSO reductase family might be required to serve as a platform for the modification of bis-MGD by sulfuration before the modified cofactor is inserted into the target enzyme. One of the enzymes of the DMSO reductase family in E. coli that has not been characterized was the protein YdhV. During the funding period, we purified and characterized the YdhV protein and we were able to identify bis-Mo-MPT as a novel form of Moco to be present in YdhV. We were able to purify YdhV both in its molybdenum and tungsten-bound form. While the physiological role of YdhV for E. coli still remains a mystery, we think that molybdenum is the metal that is specifically inserted into the enzyme.
Publications
- (2015) Assembly and catalysis of molybdenum or tungsten-containing formate dehydrogenases from bacteria. Biochimica et Biophysica Acta, 1854, 1090-1100
Hartmann, T., Schwanhold, N., Leimkühler, S.
(See online at https://doi.org/10.1016/j.bbapap.2014.12.006) - (2015) The biosynthesis of the molybdenum cofactors. J Biol Inorg Chem. 20:337-47
Mendel RR, Leimkühler S
(See online at https://doi.org/10.1155/2014/808569) - (2015) The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria.Biochim Biophys Acta 1853:1335-49
Yokoyama K, Leimkühler S
(See online at https://doi.org/10.1016/j.bbamcr.2014.09.021) - (2016) Bacterial molybdoenzymes: old enzymes for new purposes. FEMS Microbiol Rev 40: 1-18
Leimkühler S, Iobbi-Nivol C
(See online at https://doi.org/10.1093/femsre/fuv043) - (2017) Shared function and moonlighting proteins in molybdenum cofactor biosynthesis. Biol.Chem 398:1009-1026
Leimkühler S.
(See online at https://doi.org/10.1515/hsz-2017-0110)
