X-RAY CRYSTALLOGRAPHIC AND 2-DIMENSIONAL NMR INVESTIGATIONS OF A COENZYME-B12 ANALOG WITH 5'-DEOXYADENOSINE REPLACED BY 9-(CH2)3-ADENINE

The structure of (adeninylpropyl)cobalamin (AdePrCbl), a coenzyme B12 ((5'-deoxyadenosyl)cobalamin) analogue in which the ribose moiety of the adenosyl group has been replaced by a propylene chain, has been determined by X-ray diffraction methods. AdePrCbl crystallizes in the orthorhombic space group P2(1)2(1)2(1), with Z = 4, a = 23.868 (9) angstrom, b = 21.027 (7) angstrom, c = 16.047 (4) angstrom, and v = 8053.07 angstrom3. The final R value is 0.100 based on 6621 observed reflections. The general conformations of the corrin ring, benzimidazole, phosphate, and ribose in AdePrCbl are very similar to those of (5'-deoxyadenosyl)cobalamin and methylcobalamin except for the amide side chains, which show some variability in the oriantations of their amide groups. The adenine ring in AdePrCbl lies over the D ring of the corrin system, rotated about 120-degrees clockwise from its position in coenzyme B-12. The ten water molecules in the crystal structure of AdePrCbl are well located and show no evidence of disorder. Complete H-1 and C-13 NMR assignments of AdePrCbl have been made by using the following two-dimensional NMR methods: homonuclear Hartmann-Hahn spectroscopy (HOHAHA), rotating frame Overhauser enhancement spectroscopy (ROESY), H-1-detected heteronuclear multiple-quantum-coherence (HMQC) spectroscopy, and H-1-detected multiple-bond heteronuclear multiple-quantum-coherence spectroscopy (HMBC). In addition to the adenine orientation found in the crystal structure, a second orientation, in which the adenine lies over the B ring of the corrin, is suggested by H-1 NOEs and by a comparison of the H-1 and C-13 shifts AdePrCbl to those of coenzyme B-12. Our results suggest that alkyladenine groups in cobalamins may have a highly fluxional character permitting several orientations of the adenine. Previous studies have shown that binding to the B-12-dependent enzymes ribonucleotide reductase and diol dehydrase is tighter for (adeninylpentyl)cobalamin than for coenzyme B-12 and the other (adeninylalkyl)cobalamins. On the basis of our studies, we conclude that the flexibility of the alkyl chain, exhibit by the fluxional character of the alkyladenine group, and the orientation of the adenine ring could be responsible for the increased affinity of this analogue for the enzyme. Difference in the orientation of the adenine and the fluxional character of the alkyladenine group, in addition to corrin ring flexibility, may also be useful in explaining the changes in the circular dichroism spectra of (adeninylalkyl)cobalamins upon binding to ethanolamine ammonia-lyase.