THE MOLECULAR-STRUCTURE OF UDP-GALACTOSE 4-EPIMERASE FROM ESCHERICHIA-COLI DETERMINED AT 2.5 A-RESOLUTION
THE MOLECULAR-STRUCTURE OF UDP-GALACTOSE 4-EPIMERASE FROM ESCHERICHIA-COLI DETERMINED AT 2.5 A-RESOLUTION
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DOI：
10.1002/prot.340120409
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发表时间：
1992-04-01
期刊：
影响因子：
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通讯作者：
HOLDEN, HM
中科院分区：
文献类型：
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作者：
BAUER, AJ;RAYMENT, I;HOLDEN, HM
在正常的半乳糖代谢过程中，udp -半乳糖4-异丙基酶催化udp -半乳糖转化为udp -葡萄糖。来自大肠杆菌的udp -半乳糖4- epimase的分子结构现在已经被解决到名义分辨率为2.5埃。该分子从大肠杆菌中分离出来，是化学上相同亚基的二聚体，总分子量为79,000。用于本研究的酶晶体与底物类似物udp -苯生长为配合物，属于P2(1)2(1)2(1)空间群，单位胞尺寸为a = 76.3埃，b = 83.1埃，c = 132.1埃，每个不对称单元有一个二聚体。通过六个重原子衍生物的多重同构置换、分子平均和溶剂平坦化，获得了可解释的2.5埃分辨率的电子密度图。酶的每个亚基分为两个结构域。较大的n端结构域由氨基酸残基1-180组成，显示出典型的NAD+结合基序，在α螺旋的两侧有7股平行的β褶片。-折叠片的第七链是由来自较小结构域的氨基酸残基组成的。此外，这个较小的c端结构域由氨基酸残基181-338组成，包含三股β -褶片，两个主要的α -螺旋和一个螺旋转。底物类似物udp -苯结合在两个结构域之间的间隙中，其苯基环靠近NAD+的烟酰胺环。与大量的生化文献表明，每个功能二聚体只结合一个NAD+相反，该图谱清楚地显示了在二聚体中对称相关位置结合的两个烟酰胺辅因子的电子密度。同样，二聚体中的每个亚基也结合一种底物类似物。
UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP-glucose during normal galactose metabolism. The molecular structure of UDP-galactose 4-epimerase from Escherichia coli has now been solved to a nominal resolution of 2.5 angstrom. As isolated from E. coli, the molecule is a dimer of chemically identical subunits with a total molecular weight of 79,000. Crystals of the enzyme used for this investigation were grown as a complex with the substrate analogue, UDP-benzene, and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 76.3 angstrom, b = 83.1 angstrom, c = 132.1 angstrom, and one dimer per asymmetric unit. An interpretable electron density map calculated to 2.5 angstrom resolution was obtained by a combination of multiple isomorphous replacement with six heavy atom derivatives, molecular averaging, and solvent flattening.Each subunit of epimerase is divided into two domains. The larger N-terminal domain, composed of amino acid residues 1-180, shows a classic NAD+ binding motif with seven strands of parallel beta-pleated sheet flanked on either side of alpha-helices. The seventh strand of the beta-pleated sheet is contributed by amino acid residues from the smaller domain. In addition, this smaller C-terminal domain, consisting of amino acid residues 181-338, contains three strands of beta-pleated sheet, two major alpha-helices and one helical turn. The substrate analogue, UDP-benzene, binds in the cleft located between the two domains with its phenyl ring in close proximity to the nicotinamide ring of NAD+. Contrary to the extensive biochemical literature suggesting that epimerase binds only one NAD+ per functional dimer, the map clearly shows electron density for two nicotinamide cofactors binding in symmetry-related positions in the dimer. Likewise, each subunit in the dimer also binds one substrate analogue.