Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5′-deoxyadenosyl radical (5′-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe–C5′ bond between 5′-dAdo· and the [4Fe–4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5′-dAdo· intermediate. Liberation of a reactive 5′-dAdo· intermediate via homolytic metal–carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.
An RNA aptamer containing a 15-nt binding site shows high affinity and specificity for the bronchodilator theophylline. A variety of base modifications or 29 deoxyribose substitutions in binding-site residues were tested for theophyllinebinding affinity and the results were compared with the previously determined three-dimensional structure of the RNA-theophylline complex. The RNA-theophylline complex contains a U6-A28-U23 base triple, and disruption of this A28-U23 Hoogsteen-pair by a 7-deaza, 29-deoxy A28 mutant reduces theophylline binding .45-fold at 25 8C. U24 is part of a U-turn in the core of the RNA, and disruption of this U-turn motif by a 29-deoxy substitution of U24 also reduces theophylline binding by .90-fold. Several mutations outside the "conserved core" of the RNA aptamer showed reduced binding affinity, and these effects could be rationalized by comparison with the three-dimensional structure of the complex. Divalent ions are absolutely required for high-affinity theophylline binding. High-affinity binding was observed with 5 mM Mg 21 , Mn 21 , or Co 21 ions, whereas little or no significant binding was observed for other divalent or lanthanide ions. A metal-binding site in the core of the complex was revealed by paramagnetic Mn 21 -induced broadening of specific RNA resonances in the NMR spectra. When caffeine is added to the aptamer in tenfold excess, the NMR spectra show no evidence for binding in the conserved core and instead the drug stacks on the terminal helix. The lack of interaction between caffeine and the theophylline-binding site emphasizes the extreme molecular discrimination of this RNA aptamer.
A series of 9,lO-phenanthrenequinone diimine (phi) complexes of rhodium have been prepared so as to systematically explore contributions of hydrogen bonding and van der Waals interactions to DNA site specificity. The novel, synthetic complexes A-and A-[Rh(en)2phiI3+ (2), as well as the analogs [Rh(NH3)4phi]3+ (l), [Rh( [ 12]aneN4)-phi]3+ (3), and [Rh ([ 12]aneS4)phiI3+ (4) (en = ethylenediamine, [ 12]aneN4 = 1,4,7,1O-tetraazacyclodcdecane, [ 121-aneS4 = 1,4,7,1O-tetrathiacyclododecane) bind in the major groove of DNA via intercalation and promote DNA strand cleavage upon activation with UV light. Complexes 1, A-2, and 3, all of which contain axial amines, display a high sequence preference for 5'-GC-3' steps which is not observed for 4. The 5'-GC-3' preference is attributed to the hydrogen-bonding interactions between the amine ligands and the guanine 0 6 atoms in the major groove. Complex 4, which lacks hydrogen bond donating groups in the axial positions, shows instead a high degree of specificity for a S-ATG-3'site which is best explained by shape selection. 8-2 cleaves DNA with lower sequence selectivity but cleaves enantioselectively at 5'-TX-3' steps. Photocleavage of an oligonucleotide containing the substitution of 5'-UA-3' for 5'-TA-3' shows no similar enantioselectivity, and hence chiral recognition of the 5'-TA-3' step is attributed to van der Waals interactions between the methylene groups of the A-isomer and the thymine methyl groups in the major groove of DNA. These results provide a first example of binding and photoactivated cleavage using saturated amines and macrocyclic thioethers as ancillary ligands in DNA recognition by metallointercalators and illustrate how discrete elements of molecular recognition may direct specificity for a DNA site.
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