Coenzyme B induced coordination of coenzyme M via its thiol group to Ni(I) of F430 in active methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the reaction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. At the active site, it contains the nickel porphinoid F430, which has to be in the Ni(I) oxidation state for the enzyme to be active. How the substrates interact with the active site Ni(I) has remained elusive. We report here that coenzyme M (HS-CoM), which is a reversible competitive inhibitor to methyl-coenzyme M, interacts with its thiol group with the Ni(I) and that for interaction the simultaneous presence of coenzyme B is required. The evidence is based on X-band continuous wave EPR and Q-band hyperfine sublevel correlation spectroscopy of MCR in the red2 state induced with 33S-labeled coenzyme M and unlabeled coenzyme B.     

Pyranosyl-RNA (‘p-RNA’): Base-pairing selectivity and potential to replicate. Preliminary communication

Base pairing in p-RNA (β-D -ribopyranosyl-(4′ → 2′)-oligonucleotides) is not only stronger than in DNA and RNA, but also more selective in the sense that it is strictly confined to the Watson-Crick mode. Homopurine sequences (tested up to decamers) exist as single strands under conditions where they undergo reverse-Hoogsteen self-pairing in homo-DNA or Hoogsteen self-pairing in DNA. This exceptional pairing selectivity is rationalized as hinging on two structural features of p-RNA: the large inclination between backbone axis and base-pair axes in p-RNA duplexes, and the higher rigidity of the p-RNA backbone compared with RNA, DNA, and homo-DNA. The most important consequence of the pairing selectivity refers to the potential of p-RNA to replicate. Replicative copying of sequence information by nonenzymatic template-controlled ligation is not hampered by self-pairing of guanine-rich templates, as it is known to be the case in the RNA series. We have demonstrated two replicative cycles in which G-rich p-RNA-octamer templates induce sequence-selective ligation of tetramer-2′-phosphate derivatives to complementary C-rich octamer sequences, and in which the latter, with comparable efficiency, induce corresponding ligation reactions back to the original G-rich octamers. Ligation is most satisfactorily achieved after pre-activation of the 2′-phosphate groups as 2′,3′-cyclophosphate derivatives; in this version, the process does not proceed as oligocondensation, but as a genuine oligomerization. This is of considerable promise for the search for potentially natural conditions under which homochiral p-RNA strands might self-assemble and self-replicate.     

The Effect of Backbone‐Heteroatom Substitution on the Folding of Peptides – A Single Fluorine Substituent Prevents a β‐Heptapeptide from Folding into a 314‐Helix (NMR Analysis)

The β‐heptapeptides H‐βhVal‐βhAla‐βhLeu‐βhAla(X_n)‐βhVal‐βhAla‐βhLeu‐OH 3 – 7 with central 3‐amino‐2‐fluoro‐, 3‐amino‐2,2‐difluoro‐, or 3‐amino‐2‐hydroxybutanoic acid residues (βhAla(X_n)) of like and unlike configuration were subjected to a detailed NMR analysis in MeOH solution. For the geminal difluoro and for the F‐ and OH‐substituted derivatives of u‐configuration (see 5, 4 , and 7 , resp.), 14‐helices were found, i.e., with axial disposition of the hetero atoms on the helix. The two compounds containing the central l‐configured β‐amino acid moieties (see 3 and 6 ) are not helical over the full lengths of the chains; they have ‘quasi‐helical’ termini and a central turn consisting of a ten‐membered H‐bonded ring (Fig. 2, d and e). Quantum‐mechanical calculations with l‐ and u‐AcNH‐CHMe‐CHF‐CONH₂ confirm the observed preference for a conformation with antiperiplanar arrangement of the F? C and the C?O bond. The calculated energy difference between the observed ‘non‐helical’ geometry of this moiety and a hypothetical helical one is 6.4 kcal/mol (Fig. 3).     

On RNA Triplet Interactions: NMR Study of the Short Intramolecular Duplex Formed by r[GCAm1G‐p‐O(CH2CH2O)6‐p‐UGCC], Preliminary Communication

The flexible hexaethylene‐glycol linker enhances the stability of the duplex between the two tetranucleotides in compound 1 sufficiently to allow determination of the solution structure by NMR. At 4.8°, two of the three possible imino NH protons are detected as sharp signals and establish the presence of two G⋅C Watson‐Crick base pairs. Through assignment of all but one of the non‐labile protons and measurement of ¹H,¹H and ¹H,³¹P coupling constants, as well as NOEs of labile and non‐labile protons, it was possible for the first time to derive detailed structural information on such a short duplex. It forms an A‐type double helix over the full length, including the dangling nucleotides. Small variations of coupling constants and a broadening of the H−C(8) signal of m¹G4 indicate that the two nucleotides connected to the linker are conformationally slightly distorted and/or more flexible than the unlinked end of the duplex.     

On the Early Steps of Cineol Biosynthesis in Eucalyptus globulus

Samples of [4‐²H₁]‐1‐deoxyxylulose ( 17a ) and [2‐¹³C, 4‐²H₁]‐1‐deoxyxylulose ( 17b ), have been prepared by modification of known procedures and fed in aqueous solution to twiglets of Eucalyptus globulus. The probes of cineol ( 6 ) isolated from these experiments were analyzed by GC/MS, ²H‐ and ¹³C‐NMR techniques. In the experiments with 17b , the formation of five isotopomers of 6 could be detected. Their structure and relative abundance demonstrate that the ¹³C‐label is incorporated to the same extent into the two C₅‐units of 6 , and that the ²H label is retained to an extent of 57% in the starter dimethylallyl‐diphosphate unit (DMAPP; 12 ), but completely or almost completely lost in the unit derived from isopentenyl diphosphate (IPP; 11 ), in the elongation step which leads to geranyl diphosphate (GPP; 1 ). These results confirm that the recently discovered mevalonate‐independent pathway to IPP and DMAPP is operative in the biosynthesis of cineol, and indicate, together with previous finding, that, within this pathway, formation of IPP and DMAPP occurs in independent rather than in sequential steps. In addition, the demonstration of different metabolic origins for the olefinic H‐atoms of GPP ( 1 ), the aliphatic C₁₀‐precursor of 6 , paves the way for a realistic interpretation of the strikingly consistent but hitherto unexplained anomalies detected in the natural‐abundance ²H‐NMR spectra of (+)‐ and (−)‐α‐pinene and of (+)‐limonene.     

Temperature-Dependent NMR and CD Spectra of β-Peptides: On the Thermal Stability of β-Peptide Helices – Is the Folding Process of β-Peptides Non-cooperative?

Temperature-dependent NMR and CD spectra of methanol solutions of a β-hexapeptide and of a β-heptapeptide at temperatures between 298 and 393 K are reported. They establish the fact that the 3₁₄-helical secondary structures of the two β-peptides, 1 and 2 , do not `melt' in the temperature range investigated. This is in sharp contrast to the behavior of the helices of α-peptides and proteins which undergo cooperative unfolding (`denaturing') upon heating. A non-cooperative mechanism is proposed, with a stepwise, rather than an `un-zipping' opening of H-bonded rings (cf. Fig. 6). The experimental results are regarded as evidence that, of the three effects which have been identified as contributing to the stability of β-peptide helices, i.e., H-bonding, hydrophobic interactions, and ethane staggering, the latter one is predominant.     

NMR Solution Structure of the Duplex Formed by Self‐Pairing of α‐L‐Arabinopyranosyl‐(4′2′)‐(CGAATTCG)

The solution structure of the duplex formed by α‐L ‐arabinopyranosyl‐(4′→2′)‐(CGAATTCG) was studied by NMR. The resonances of all H‐, P‐ and most C‐atoms could be assigned. Dihedral angles and distance estimates derived from coupling constants and NOESY spectra were used as restraints in a simulated annealing calculation, which generated a well‐defined bundle of structures for the six innermost nucleotide pairs. The essential features of the resulting structures are an antiparallel, Watson Crick‐paired duplex with a strong backbone inclination of ca. −50° and, therefore, predominant interstrand base stacking. The very similar inclination and rise parameters of arabinopyranosyl‐(4′→2′)‐oligonucleotides and p‐RNA explain why these two pentapyranosyl isomers are able to cross‐pair.     

Isotopically Labelled and Unlabelled β‐Peptides with Geminal Dimethyl Substitution in 2‐Position of Each Residue: Synthesis and NMR Investigation in Solution and in the Solid State

The preparation of (S)‐β^2,2,3‐amino acids with two Me groups in the α‐position and the side chains of Ala, Val, and Leu in the β‐position (double methylation of Boc‐β‐HAla‐OMe, Boc‐β‐Val‐OMe, and Boc‐β‐Leu‐OMe, Scheme 2) is described. These β‐amino acids and unlabelled as well as specifically ¹³C‐ and ¹⁵N‐labelled 2,2‐dimethyl‐3‐amino acid (β^2,2‐HAib) derivatives have been coupled in solution (Schemes 1, 3 and 4) to give protected (N‐Boc, C‐OMe), partially protected (N‐Boc/C‐OH, N‐H/C‐OMe), and unprotected β^2,2‐ and β^2,2,3‐hexapeptides, and β^2,2‐ and β^2,2,3‐heptapeptides 1 – 7 . NMR Analyses in solution (Tables 1 and 2, and Figs. 2–4) and in the solid state (2D‐MAS NMR measurements of the fully labelled Boc‐(β^2,2‐HAib)₆‐OMe ([¹³C₃₀, ¹⁵N₆]‐ 1e ; Fig. 5), and TEDOR/REDOR NMR investigations of mixtures (Fig. 6) of the unlabelled Ac‐(β^2,2‐HAib)₇‐OMe ( 4 ) and of a labelled derivative ([¹³C₄,¹⁵N₂]‐ 5 ; Figs. 7–11, and 19), a molecular‐modeling study (Figs. 13–15), and a search in the Cambridge Crystallographic Data Base (Fig. 16) allow the following conclusions: i) there is no evidence for folding (helix or turn) or for aggregation to sheets of the geminally dimethyl substituted peptide chains in solution; ii) there are distinct conformational preferences of the individual β^2,2‐ and β^2,2,3‐amino acid residues: close to eclipsing around the C(O) C(Me₂(CHR)) bond (τ_1,2), almost perfect staggering around the C(2) C(3) ethane bond (τ_2,3), and antiperiplanar arrangement of H(C3) and H(N) (τ_3,N; Fig. 12) in the solid state; iii) the β^2,2‐peptides may be part of a turn structure with a ten‐membered H‐bonded ring; iv) the main structure present in the solid state of F₃CCO(β^2,2‐HAib)₇‐OMe is a nonfolded chain (>30 Å between the termini and >20 Å between the N‐terminus and the CH₂ group of residue 5) with all CO bonds in a parallel alignment (±10°). With these structural parameters, a simple modelling was performed producing three (maybe four) possible chain geometries: one fully extended, two with parallel peptide planes (with zick‐zack and crankshaft‐type arrangement of the peptide bonds), and (possibly) a fourth with meander‐like winding ( D – G in Figs. 17 and 18).     

Solution structure of a DNA duplex containing a biphenyl pair

Hydrogen-bonding and stacking interactions between nucleobases are considered to be the major noncovalent interactions that stabilize the DNA and RNA double helices. In recent work we found that one or multiple biphenyl pairs, devoid of any potential for hydrogen bond formation, can be introduced into a DNA double helix without loss of duplex stability. We hypothesized that interstrand stacking interactions of the biphenyl residues maintain duplex stability. Here we present an NMR structure of the decamer duplex d(GTGACXGCAG) d(CTGCYGTCAC) that contains one such X/Y biaryl pair. X represents a 3',5'-dinitrobiphenyl- and Y a 3',4'-dimethoxybiphenyl C-nucleoside unit. The experimentally determined solution structure shows a B-DNA duplex with a slight kink at the site of modification. The biphenyl groups are intercalated side by side as a pair between the natural base pairs and are stacked head to tail in van der Waals contact with each other. The first phenyl rings of the biphenyl units each show tight intrastrand stacking to their natural base neighbors on the 3'-side, thus strongly favoring one of two possible interstrand intercalation structures. In order to accommodate the biphenyl units in the duplex the helical pitch is widened while the helical twist at the site of modification is reduced. Interestingly, the biphenyl rings are not static in the duplex but are in dynamic motion even at 294 K.