Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (ϳ70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein.A cetogenic bacteria were the first anaerobes described to utilize phenyl methyl ethers as energy substrates (2). These organisms mediate the cleavage of the substrate ether bond and utilize the methyl group, which is oxidized to CO 2 in the oxidative part of catabolism. The reducing equivalents derived from methyl group oxidation are transferred to CO 2 upon the formation of an enzyme-bound carbon monoxide, which is then combined with further methyl groups to finally yield acetate (6). The key enzymes in the methylotrophic phenyl methyl ether metabolism of acetogens such as Acetobacterium dehalogenans (23) and Moorella thermoacetica (13) are the O-demethylases. These inducible enzyme systems mediate the ether cleavage and transfer of the methyl group to tetrahydrofolate (FH 4 ). Until now, three of these acetogenic O-demethylase systems were purified and characterized (7,14,25). In general, they consist of four protein components: two methyltransferases (MTs) (MT I and MT II), a corrinoid protein (CP), and an activating enzyme (AE). Both MTs and CP are involved in the catalytic cycle (Fig. 1) (Fig. 1). In A. dehalogenans, the genes encoding MT I, MT II, and CP are usually organized into an operon (31). Only one AE gene has been detected so far; this gene is not part of a...
Proteomic biomarker search requires the greatest analytical reproducibility and detailed information on altered proteoforms. Our protein pre-fractionation applies orthogonal native chromatography and conserves important features of protein variants such as native molecular weight, charge and major glycans. Moreover, we maximized reproducibility of sample pre-fractionation and preparation before mass spectrometry by parallelization and automation. In blood plasma and cerebrospinal fluid (CSF), most proteins, including candidate biomarkers, distribute into a multitude of chromatographic clusters. Plasma albumin, for example, divides into 15-17 clusters. As an example of our technique, we analyzed these albumin clusters from healthy volunteers and from dogs and identified cluster-typical modification patterns. Renal disease further modifies these patterns. In human CSF, we found only a subset of proteoforms with fewer modifications than in plasma. We infer from this example that our method can be used to identify and characterize distinct proteoforms and, optionally, enrich them, thereby yielding the characteristics of proteoform-selective biomarkers.
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