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Abstract: The organometallic complex coenzyme B12 (adenosyl cobalamin, AdoCbl) is not only an essential coenzyme in many biochemical reactions of most if not all living organisms but has lately been shown to play a crucial role in the regulation of B12 related genes. As a consequence, coenzyme B12 has been a target of intense research. However, the investigations of AdoCbl have often been hampered due to its high light-sensitivity leading to decomposition of the compound within a few seconds. Here, we describe a strategy to synthesize more light-stable coenzyme B12 analogs, which show similar steric properties as adenosyl cobalamin. The synthesis, structural characterization as well as the pH dependent “base-on/base-off” behavior of cyanide bridged vitamin B12 conjugates with either a cis-[(NH3)2Pt]2+ or an [enPt]2+ moiety, leading to cis-[(NH3)2PtCl-vitB12]+ (1) and [enPtCl-vitB12]+ (2) are reported. The subsequent reaction of cis-[(NH3)2PtCl-vitB12]+ with the model nucleobase 9-methyladenine leads to the corresponding adduct, where the adenine moiety is coordinated to the Pt2+ center either via N1 or N7. This compound is light-stable and harbors the adenine moiety in the same distance of 5Å above the corrin plane as present in the highly light-sensitive adenosyl cobalamin. [Copyright &y& Elsevier]

Riboswitches are structural elements of mRNA involved in the regulation of gene expression by responding to specific cellular metabolites. To fulfil their regulatory function, riboswitches prefold into an active state, the so-called binding competent form, that guarantees metabolite binding and allows a consecutive refolding of the RNA. Here, we describe the folding pathway to the binding competent form as well as the ligand free structure of the moaA riboswitch of E. coli. This RNA proposedly responds to the molybdenum cofactor (Moco), a highly oxygen-sensitive metabolite, essential in the carbon and sulfur cycles of eukaryotes. K+- and Mg2+-dependent footprinting assays and spectroscopic investigations show a high degree of structure formation of this RNA already at very low ion-concentrations. Mg2+ facilitates additionally a general compaction of the riboswitch towards its proposed active structure. We show that this fold agrees with the earlier suggested secondary structure which included also a long-range tetraloop/tetraloop-receptor like interaction. Metal ion cleavage assays revealed specific Mg2+-binding pockets within the moaA riboswitch. These Mg2+ binding pockets are good indicators for the potential Moco binding site, since in riboswitches, Mg2+ was shown to be necessary to bind phosphate-carrying metabolites. The importance of the phosphate and of other functional groups of Moco is highlighted by binding assays with tetrahydrobiopterin, the reduced and oxygen-sensitive core moiety of Moco. We demonstrate that the general molecular shape of pterin by its own is insufficient for the recognition by the riboswitch. The metal-ion dependent folding pathway of the ligand-free moaA riboswitch was studied towards the formation of the active, molybdenum cofactor-binding competent fold. This fold was further characterized by various footprinting methods while Tb(III) cleavage identified specific Mg2+ binding sites. Binding experiments with pterins complete this study. [Display omitted] • The metal-ion dependent folding of a molybdenum cofactor riboswitch is described. • The binding competent fold forms at very low Mg2+-concentrations. • A tetraloop/tetraloop-receptor like interaction stabilizes the tertiary structure. • Specific Mg2+ binding sites were identified. • The pterin moiety of the molybdenum cofactor is not sufficient for recognition/refolding of the riboswitch. [ABSTRACT FROM AUTHOR]