STRUCTURE ANALYSIS OF B12 BINDING SUBUNIT OF GLUTAMATE MUTASE

谷氨酸变位酶B12结合亚基的结构分析

• 批准号: 6281283
• 负责人: MARTHA L LUDWIG
• 金额: $ 0.46万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-15 至 1999-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6281283
• 关键词: biological products; biomedical resource; proteins; radiography; structural biology

Coenzyme B12-dependent glutamate mutase catalyzes the isomerization of (S)-glutamate to (2S, 3S)-3-methylaspartate, the first step in glutamate fermentation by Clostridium tetanomorphum . To better understand the mechanism of glutamate mutase, and in particular how the apoprotein modulates the reactivity of the B12 cofactor, we have crystallized the B12-binding subunit of glutamate mutase (15 kDa), in the presence of coenzyme B12 (AdoCbl). Crystals as large as 1.2 mm x 0.8 mm x 0.7 mm have been obtained routinely, and some of them diffract to a resolution of 2.2
. All data sets have indexed as primitive tetragonal with unit cell dimensions a = b = 104.7
, c = 162.7
. The large unit cell indicates that the asymmetric unit likely contains four to six molecules. A novel approach to MAD phasing, employing the intrinsic cobalt of AdoCbl as an anomalous scatterer, was recently conducted at CHESS. Two data sets (91% complete to 2.6
) taken at energies corresponding to the peak and inflection points of cobalt were combined with data taken on a home source (1.5418
radiation, used as a remote wavelength). Four cobalt sites were identified in anomalous difference Patterson maps. Electron density maps generated from phases calculated by SOLVE have not been interpretable. However, the combination of phasing information from the cobalt experiments with isomorphous replacement phasing from a recently identified mercury derivative may prove adequate for structure determination.

辅酶B12依赖性谷氨酸盐催化 （S）-谷氨酸异构化为（2S，3S）-3-甲基天冬氨酸， 破伤风梭菌谷氨酸发酵的第一步。 为了更好地了解谷氨酸盐代谢的机制， 特别是载脂蛋白如何调节B12的反应性， 辅因子，我们已经结晶的B12结合亚基谷氨酸 在辅酶B12（B12 Cbl）存在下，将该酶（15 kDa）与酶（15 kDa）反应。 晶体 常规获得的尺寸为1.2 mm x 0.8 mm x 0.7 mm， 其中一些分辨率为2.2
.所有数据集都有 索引为具有晶胞尺寸a = B = 104.7
，c = 162.7
.大的晶胞表明， 不对称单元可能含有4到6个分子。 一种新型 MAD定相的方法，采用C10 Cbl的固有钴作为 一个异常散射体，最近在国际象棋进行。 两个数据 （91%完成至2.6
），其能量对应于 将钴的峰值和拐点与以下数据相结合： 一个家庭来源（1.5418
辐射，用作远程波长）。 四 钴网站确定异常差异帕特森地图。 由SOLVE计算的相位生成的电子密度图具有 无法解释。 然而，相结合的阶段 来自同晶置换钴实验的信息 从最近发现的汞衍生物中逐步淘汰， 适合结构测定。