Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) complexed with GMP: evidence for a substrate-induced transferase active site.
Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) complexed with GMP: evidence for a substrate-induced transferase active site.
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腺苷酰胺激酶/腺苷磷酸鸟苷基转移酶 (CobU) 与 GMP 复合的三维结构：底物诱导转移酶活性位点的证据。
DOI：
10.1021/bi990910x
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Rayment,I
中科院分区：
文献类型：
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作者：
Thompson,TB;Thomas,MG;Escalante-Semerena,JC;Rayment,I
本文用分子置换法测定了鼠伤寒沙门氏菌腺苷可宾酰胺激酶/腺苷可宾酰胺磷酸鸟苷酰转移酶（CobU）与GMP结合的X-射线晶体结构。CobU是一种双功能酶，其催化腺苷二酰胺环的1-氨基-O-2-丙醇侧链的磷酸化，随后作为鸟苷酰转移酶发挥作用以形成腺苷二酰胺·GDP。转移酶活性涉及共价酶-鸟苷酰中间体，其最可能是与His 46的氨基磷酸酯连接。以前的研究表明，该酶是一个同源三聚体，并采用风车形状。每个亚基由六条平行β链和一条反平行链的单个结构域组成，两侧共有五个α螺旋和一个螺旋转角。有趣的是，脱辅基酶中的His 46位于离激酶活性位点或P环基序相当远的距离处，并且是溶剂暴露的[Thompson，T. B.，等人（1998）Biochemistry 37，7686 - 7695]。为了研究两个活性位点的结构关系，将CobU与GTP和焦磷酸盐共结晶。晶体属于空间群P212121，具有以下晶胞尺寸：a= 58.4，B= 87.8，和c = 101.6。该结构显示水解产物GMP的电子密度，而不是预期的共价鸟苷酰中间体，其似乎已在晶格中水解。即便如此，CobU表现出实质性的构象重排。含有His 46（鸟苷酰化位点）的螺旋轴相对于载脂蛋白结构旋转30°并平移11 °，并伴随着螺旋末端的补偿性解旋和重绕，以允许在β-链2和α-螺旋2之间诱导鸟苷结合口袋。这种构象变化使His 46的Cα与P-环基序接近约10 μ m，使得位于P-环中的磷酸根离子与GMP的α-磷酸根仅相距6 μ m。这表明，P-环基序可用于协调的转移酶和激酶反应中的末端磷酸，并意味着两个反应的活性位点重叠。
The X-ray crystal structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) fromSalmonella typhimuriumbound to GMP has been determined by molecular replacement to 2.2 Å resolution. CobU is a bifunctional enzyme, which catalyzes the phosphorylation of the 1-amino-O-2-propanol side chain of the adenosylcobinamide ring and subsequently functions as a guanylyltransferase to form adenosylcobinamide·GDP. The transferase activity involves a covalent enzyme−guanylyl intermediate that is most likely a phosphoramidate linkage to His46. Previous studies have shown that the enzyme is a homotrimer and adopts a pinwheel shape. Each subunit consists of a single domain of six parallel β-strands and one antiparallel strand flanked on either side by a total of five α-helices and one helical turn. Interestingly, His46in the apoenzyme is located a considerable distance from the kinase active site or P-loop motif and is solvent-exposed [Thompson, T. B., et al. (1998)Biochemistry37, 7686−7695]. To examine the structural relationship of the two active sites, CobU was cocrystallized with GTP and pyrophosphate. Crystals belong to space groupP212121with the following unit cell dimensions:a= 58.4 Å,b= 87.8 Å, andc= 101.6 Å. The structure shows electron density for the hydrolysis product GMP rather than the expected covalent guanylyl intermediate which appears to have been hydrolyzed in the crystal lattice. Even so, CobU exhibits a substantial conformational rearrangement. The helix axis containing His46, the site of guanylylation, rotates 30° and translates 11 Å relative to the apo structure and is accompanied by compensatory unwinding and rewinding at the helix ends to allow the induction of a guanosine binding pocket between β-strand 2 and α-helix 2. This conformational change brings the Cαof His46approximately 10 Å closer to the P-loop motif such that a phosphate ion located in the P-loop is only 6 Å from the α-phosphate of GMP. This suggests that the P-loop motif may be used to coordinate the terminal phosphates in both the transferase and kinase reactions and implies that the active sites for both reactions overlap.
R.G.Duggleby：“酶学方法”
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钴胺素生物合成的生物化学和分子遗传学。
DOI：
10.1016/s0079-6603(08)61010-7
发表时间：
1997
期刊：
Progress in nucleic acid research and molecular biology
影响因子：
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作者：
Rondon,MR;Trzebiatowski,JR;Escalante-Semerena,JC
通讯作者：
Escalante-Semerena,JC