EC Number   |
Reference |
|---|
   4.3.1.7 | deletion mutants of the enzyme beta subunit DELTA4-30 and DELTA4-43, to 8.0 and 2.1 A resolution, respcetively |
713644 |
| 4.3.1.7 | For crystallization trials, protein sample is dialyzed against 50 mM HEPES with pH 7.0 and concentrates to a final concentration of about 1 mg/ml. Purification results in highly pure protein that crystallizes readily under many different conditions, protein forms thin hexagonal plate-shaped crystals belonging to space group P3. Best crystals of Eut-L_NHIS are obtained in 3.3 M ammonium acetate, 5% polyethylene glycol 400 and 50 mM Tris buffer pH 7.5, crystals grow as hexagonal plates. Eut-L_CHIS crystals grow as single hexagonal plates with sharp edges. Crystals grow in 2 M NaCl, 100 mM phosphate, 100 mM MES buffer pH 6.5 and 4% PEG 400. |
690280 |
| 4.3.1.7 | N-terminal His6-tagged beta subunit lacking residues Lysbeta4-Cysbeta43, complexed with CN-cobalamin and (R)-2-amino-1-propanol or (S)-2-amino-1-propanol. The lower affinity for the (R)-enantiomer may be due to the conformational change of the ValR326 side chain of the enzyme. The pro-S hydrogen atom on C1 is abstracted by the adenosyl radical from both enantiomeric substrates. The NH2 group migrates from C2 to C1 by a suprafacial shift, with inversion of configuration at C1 for both enantiomeric substrates. (R)-2-amino-1-propanol is deaminated by the enzyme with inversion of configuration at C2, whereas the (S)-enantiomer is deaminated with retention. The rotameric radical intermediate from the (S)-enantiomer undergoes flipping to the rotamer from the (R)-enantiomer before the hydrogen back-abstraction, suggesting the preference of the enzyme active site for the rotamer from the (R)-enantiomer in equilibration, partly explained by steric repulsion of the (S)-enantiomer-derived product radical at C3 with the PheR329 and LeuR402 residues |
714261 |
| 4.3.1.7 | N-terminal His6-tagged beta subunit lacking residues Lysbeta4-Cysbeta43, in complex with cyanocobalamin and in complex with cyanocobalamin or adeninylpentylcobalamin and substrates. The enzyme exists as a trimer of the (alphabeta)2 dimer. The active site is in the (beta/alpha)8 barrel of the-subunit, the beta-subunit covers the lower part of the cobalamin that is bound in the interface of the alpha- and beta-subunits. The structure complexed with adeninylpentylcobalamin reveals the presence of an adenine ring-binding pocket in the enzyme that accommodates the adenine moiety through a hydrogen bond network. The substrate is bound by six hydrogen bonds with active-site residues. Arg160 contributes to substrate binding most likely by hydrogen bonding with the O1 atom. Marked angular strains and tensile forces induced by tight enzyme-coenzyme interactions are responsible for breaking the coenzyme-Co-C bond. A major structural change upon substrate binding is not observed with this particular enzyme. Glu287, one of the substrate-binding residues, has a direct contact with the ribose group of the modeled adenosylcobalamin, which may contribute to the substrate-induced additional labilization of the Co-C bond |
715530 |
