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The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical(More)
Infrared-spectroscopic studies on the [NiFe]-hydrogenase of Chromatium vinosum-enriched in 15N or 13C, as well as chemical analyses, show that this enzyme contains three non-exchangeable, intrinsic, diatomic molecules as ligands to the active site, one carbon monoxide molecule and two cyanide groups. The results form an explanation for the three non-protein(More)
BACKGROUND This study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain(More)
[NiFe] hydrogenases catalyze the reversible heterolytic cleavage of molecular hydrogen. Several oxidized, inactive states of these enzymes are known that are distinguishable by their very different activation properties. So far, the structural basis for this difference has not been understood because of lack of relevant crystallographic data. Here, we(More)
The incorporation of carbon into the carbon monoxide and cyanide ligands of [NiFe]-hydrogenases has been investigated by using (13)C labelling in infrared studies of the Allochromatium vinosum enzyme and by (14)C labelling experiments with overproduced Hyp proteins from Escherichia coli. The results suggest that the biosynthetic routes of the carbon(More)
Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation. However, little is known about the contribution of translational regulation. Recently, we have developed a pulse labeling method using the methionine analog azidohomoalanine to determine the relative amounts of(More)
The iron-sulfur-cluster-free hydrogenase Hmd (H(2)-forming methylenetetrahydromethanopterin dehydrogenase) from methanogenic archaea has recently been found to contain one iron associated tightly with an extractable cofactor of yet unknown structure. We report here that Hmd contains intrinsic CO bound to the Fe. Chemical analysis of Hmd revealed the(More)
Cross-links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross-links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross-linkers that enable isolation of the cleavage products while preserving information(More)
In [FeFe]-hydrogenases, the H cluster (hydrogen-activating cluster) contains a di-iron centre ([2Fe]H subcluster, a (L)(CO)(CN)Fe(μ-RS2)(μ-CO)Fe(CysS)(CO)(CN) group) covalently attached to a cubane iron-sulphur cluster ([4Fe-4S]H subcluster). The Cys-thiol functions as the link between one iron (called Fe1) of the [2Fe]H subcluster and one iron of the(More)