Structural and functional analysis of an l-serine O-phosphate decarboxylase involved in norcobamide biosynthesis.

Structural diversity of natural cobamides (Cbas, B12 vitamers) is limited to the nucleotide loop. The loop is connected to the cobalt-containing corrin ring via an (R)-1-aminopropan-2-ol O-2-phosphate (AP-P) linker moiety. AP-P is produced by the l-threonine O-3-phosphate (l-Thr-P) decarboxylase CobD. Here, the CobD homolog SMUL_1544 of the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans was characterized as a decarboxylase that produces ethanolamine O-phosphate (EA-P) from l-serine O-phosphate (l-Ser-P). EA-P is assumed to serve as precursor of the linker moiety of norcobamides that function as cofactors in the respiratory reductive dehalogenase. SMUL_1544 (SmCobD) is a pyridoxal-5'-phosphate (PLP)-containing enzyme. The structural analysis of the SmCobD apoprotein combined with the characterization of truncated mutant proteins uncovered a role of the SmCobD N-terminus in efficient l-Ser-P conversion.

Thermal proteome profiling allows quantitative assessment of interactions between tetrachloroethene reductive dehalogenase and trichloroethene.

Thermal proteome profiling (TPP) is increasingly applied in eukaryotes to investigate protein-ligand binding through protein melting curve shifts induced by the presence of a ligand. In anaerobic bacteria, identification of protein-substrate interactions is a major challenge. We applied TPP to Sulfurospirillum multivorans, which is able to use trichloroethene as electron acceptor for growth, to investigate the interaction of its tetrachloroethene reductive dehalogenase PceA with trichloroethene. Several modifications in the protocol (e.g., incubation under anaerobic conditions; increasing the temperature range up to 97 °C) extended the protein detection range and allowed the investigation of oxygen-sensitive proteins. Enzymatic reductive dehalogenation was prevented by omitting the electron donor during incubations. This enabled detecting the interaction of PceA with trichloroethene and confirmed that trichloroethene is a substrate of this enzyme. Interestingly, a putative response regulator showed a similar trend, which is the first biochemical hint for its proposed role in trichloroethene respiration. We proved that our TPP approach facilitates the identification of protein-substrate interactions of strictly anaerobic reductive dehalogenases and probably their regulators. This strategy can be used to identify yet unknown substrate specificities and possible signal-sensing proteins, and therefore has the potential to elucidate one of the unresolved fields in research on organohalide-respiring bacteria. SIGNIFICANCE: The assessment of enzyme-substrate or protein-ligand interactions in organohalide-respiring bacteria is a fundamental challenge. Thermal proteome profiling (TPP) allows elucidating proteome-wide thermal stability changes relying on the sensitivity of modern mass spectrometry. This gives access to the identification of interactions not detectable with other methods. In this TPP study, we demonstrate the interactions of a chlorinated substrate with a reductive dehalogenase and potentially with a response regulator, thereby supporting the response regulator's function in organohalide respiration. The strategy might also be applied to identify yet unknown substrates of other enzymes in bacteria which are difficult to investigate or for which only low amounts of biomass are available. The assessment of enzyme-substrate interactions, which might enable conclusions about enzyme specificities, represents a new application for TPP.

Selective Utilization of Benzimidazolyl-Norcobamides as Cofactors by the Tetrachloroethene Reductive Dehalogenase of Sulfurospirillum multivorans.

The organohalide-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated, and their use as cofactor by PceA was analyzed. Exogenously applied 5-methylbenzimidazole (5-MeBza), 5-hydroxybenzimidazole (5-OHBza), and 5-methoxybenzimidazole (5-OMeBza) were found to be efficiently incorporated as lower bases into norcobamides (NCbas). Structural analysis of the NCbas by nuclear magnetic resonance spectroscopy uncovered a regioselectivity in the utilization of these precursors for NCba biosynthesis. When 5-MeBza was added, a mixture of 5-MeBza-norcobamide and 6-MeBza-norcobamide was formed, and the PceA enzyme activity was affected. In the presence of 5-OHBza, almost exclusively 6-OHBza-norcobamide was produced, while in the presence of 5-OMeBza, predominantly 5-OMeBza-norcobamide was detected. Both NCbas were incorporated into PceA, and no negative effect on the PceA activity was observed. In crystal structures of PceA, both NCbas were bound in the base-off mode with the 6-OHBza and 5-OMeBza lower bases accommodated by the same solvent-exposed hydrophilic pocket that harbors the adenine as the lower base of authentic norpseudo-B12 In this study, a selective production of different norcobamide isomers containing singly substituted benzimidazoles as lower bases is shown, and unique structural insights into their utilization as cofactors by a cobamide-containing enzyme are provided.IMPORTANCE Guided biosynthesis of norcobamides containing singly substituted benzimidazoles as lower bases by the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans is reported. An unprecedented specificity in the formation of norcobamide isomers containing hydroxylated or methoxylated benzimidazoles was observed that implicated a strict regioselectivity of the norcobamide biosynthesis in the organism. In contrast to 5,6-dimethylbenzimidazolyl-norcobamide, the incorporation of singly substituted benzimidazolyl-norcobamides as a cofactor into the tetrachloroethene reductive dehalogenase was not impaired. The enzyme was found to be functional with different isomers and not limited to the use of adeninyl-norcobamide. Structural analysis of the enzyme equipped with either adeninyl- or benzimidazolyl-norcobamide cofactors visualized for the first time structurally different cobamides bound in base-off conformation to the cofactor-binding site of a cobamide-containing enzyme.

Cobamide-mediated enzymatic reductive dehalogenation via long-range electron transfer.

The capacity of metal-containing porphyrinoids to mediate reductive dehalogenation is implemented in cobamide-containing reductive dehalogenases (RDases), which serve as terminal reductases in organohalide-respiring microbes. RDases allow for the exploitation of halogenated compounds as electron acceptors. Their reaction mechanism is under debate. Here we report on substrate-enzyme interactions in a tetrachloroethene RDase (PceA) that also converts aryl halides. The shape of PceA's highly apolar active site directs binding of bromophenols at some distance from the cobalt and with the hydroxyl substituent towards the metal. A close cobalt-substrate interaction is not observed by electron paramagnetic resonance spectroscopy. Nonetheless, a halogen substituent para to the hydroxyl group is reductively eliminated and the path of the leaving halide is traced in the structure. Based on these findings, an enzymatic mechanism relying on a long-range electron transfer is concluded, which is without parallel in vitamin B12-dependent biochemistry and represents an effective mode of RDase catalysis.