Crystal structure of a predicted precorrin‐8x methylmutase from <i>Thermoplasma acidophilum</i>

Introduction.The biosynthesis of vitamin B 12 involves many enzymatic steps and two evolutionarily divergent pathways.Although the pathways differ in their requirement for molecular oxygen and the timing of cobalt insertion, the precorrin-8x methylmutase genes cobH and cbiC share greater than 30% identity, and are designated as the CobH family. 1 These enzymes catalyze the methyl isomerization of metal-free (CobH) and cobalt bound (CbiC) precorrin-8x to hydrogenobyrinic acid (HBA).We have determined the 2.1-Å crystal structure of precorrin-8x methylmutase from the facultative anaerobe Thermoplasma acidophilum (Ta0654).Although cobalamin synthesis has not been investigated in T. acidophilum, the structure bears a striking resemblance to CobH from Pseudomonas denifitricans (Pa2905), to which it shares 33% sequence identity.The structure-function relationships for Pa2905 have been characterized, including the role of catalytic residues and crystallographic observation of the product HBA in the active site formed by dimerization.[2][3][4] Methods and Results.Growth, expression, and purification of T. acidophilum precorrin-8x methylmutase (gene designation ta0654) were performed essentially as described by Korolev et al. 5 Seleno-L-methionine (SeMet)substituted protein crystallized at room temperature in 14% polyethylene glycol (PEG) 3350, 0.5 M di-ammonium tartrate, and 4% glycerol (pH ϳ6.6).The crystal belongs to the orthorhombic space group P2 1 2 1 2 1 .Multiple-wavelength anomalous dispersion (MAD) data were measured at the Structural Biology Center 191D beamline of the Advanced Photon Source (APS), Argonne National Laboratory (ANL), and were integrated and scaled using the HKL2000 suite.6 The asymmetric unit contained 4 protein polypeptides, with 9 SeMet per polypeptide.Twenty-four of the 36 Se atoms in the asymmetric unit were identified with SOLVE-2.02.7 The MAD map was then subjected to density modification using the RESOLVE subroutine in SOLVE-2.02, which allowed autotracing of most main-chain atoms for polypeptide B. Polypeptides A, C, and D were modeled using monomer B prior to refinement using the Crystallography & NMR System (CNS-1.1).8 After iterative rounds of model building in O 9 and refinement with CNS (simulated annealing, positional minimization, water molecule identification, individual isotropic B refinement), the final 2.1-Å model contains 765 protein residues and 627 waters.No noncrystallographic symmetry restraints were applied.Due to poor electron density, N-terminal residues A1-A16, B1-B13, C1-C15, D1-D15, and C-terminal residue 207 for all polypeptides were not included in the final model.According to PROCHECK, 10 90.3% of residues were in the most favored Ramachandran plot region, 9.3% were in the additionally allowed region, and 0.4% were in the generously allowed region.The final R work and R free were

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