Thomä N H, Leadlay P F
Cambridge Centre for Molecular Recognition, University of Cambridge, United Kingdom.
Protein Sci. 1996 Sep;5(9):1922-7. doi: 10.1002/pro.5560050919.
Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)-dependent methylmalonyl-CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic alpha-subunit of the heterodimeric methylmalonyl-CoA mutase from the bacterium Propionibacterium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three-dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X-ray crystal structure of the P. shermanii methylmalonyl-CoA mutase. Eight mutations are found in the C-terminal B12-binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin-like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent-exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N-terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl-CoA esters gain access to the active site (W105R). The extreme N-terminus of methylmalonyl-CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.
编码辅酶B12（腺苷钴胺素）依赖性甲基丙二酰辅酶A变位酶的人类基因中的点突变会引发一种遗传性丙酸代谢紊乱疾病，称为甲基丙二酸尿症。几乎所有此类突变都会改变人类同二聚体酶中的氨基酸，这些氨基酸与来自费氏丙酸杆菌的异二聚体甲基丙二酰辅酶A变位酶催化α亚基中的残基相同，成熟的人类酶与该细菌的酶整体序列同一性为65%。为了探究特定突变如何导致观察到的临床表型，将12个已知突变映射到人类酶亚基的三维同源模型上，该模型是使用MODELLER程序基于最近发表的费氏丙酸杆菌甲基丙二酰辅酶A变位酶2.0 Å X射线晶体结构生成的。在C末端B12结合结构域中发现了8个突变，其中4个（G623R、G626C、G630E、G703R）与咕啉直接接触，并聚集在由蛋白质提供的用于配位B12辅因子钴原子的组氨酸配体（H627）周围。在这些位置中的任何一个引入侧链，特别是带电荷的侧链，预计会破坏类黄素氧还蛋白样折叠并严重损害其与B12的结合。该结构域中另外两个高度保守的甘氨酸残基（G648D、G717V）的突变以及突变H678R中额外正电荷的引入也会破坏折叠中的关键元件。溶剂暴露环中的精氨酸突变为疏水残基（R694W）也具有致病性。其余突变已映射到变位酶的N末端区域，其中两个在亚基界面附近（A377E）或酰基辅酶A酯进入活性位点的狭窄通道附近（W105R）引入了一个埋藏的、未补偿的电荷。甲基丙二酰辅酶A变位酶的极端N末端预计会与另一个亚基广泛接触，该区域的一个突变体（R93H）可能会阻止二聚体的正确组装。
