SOLUTION STRUCTURE OF THE MUTT ENZYME, A NUCLEOSIDE TRIPHOSPHATE PYROPHOSPHOHYDROLASE
SOLUTION STRUCTURE OF THE MUTT ENZYME, A NUCLEOSIDE TRIPHOSPHATE PYROPHOSPHOHYDROLASE
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DOI：
10.1021/bi00046a006
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发表时间：
1995-11-21
期刊：
影响因子：
2.9
通讯作者：
MILDVAN, AS
中科院分区：
文献类型：
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作者：
ABEYGUNAWARDANA, C;WEBER, DJ;MILDVAN, AS
MutT酶（129个残基）通过在很少被攻击的β - p上取代，催化正常和诱变核苷三磷酸水解，如8-氧- dgtp，生成NMP和焦磷酸。先前对MutT的异核磁共振研究表明，二级结构由一个五链混合β -片组成，由环i - α -螺旋i -环II基序连接，通过两个紧转和环III，并由环iv - α -螺旋II终止[Abeygunawardana et al. (1993) Biochemistry 32, 13071-13080；Weber等人（1993）生物化学32,13081-13087)。目前已经通过3D C(CO)NH和HCCH-TOCSY实验完成了H-1和C-13共振的完整侧链分配。利用三维n -15分辨noesi - hsqc和三维c -13分辨noesi - hsqc光谱获得的1461个质子间邻近点（每个残基11个），包括372个远程NOEs，以及65个二面角约束和34个主氢键约束，利用X-PLOR程序通过距离几何、模拟退火和能量最小化来确定MutT的三级结构。这种结构是球状的，紧凑的，β -薄片的平行部分夹在两个α -螺旋之间，形成α + β折叠。必需的二价阳离子先前已被证明与Gly-37、Gly-38、Lys-39和Glu-57附近的残基结合，通过核磁共振弛豫方法，核苷酸已被证明与Leu-54和Val-58附近的残基结合[Frick et al. (1995) Biochemistry 34, 5577-5586]。MutT的三级结构表明，这些残基沿着酶的I-螺旋环I区彼此靠近。5个谷氨酸残基（41,53,56,57和98）形成一个具有强烈负静电电位的斑块，可能构成金属结合位点。该位点与β链a、C和D和环I之间的深间隙相连，这可能是核苷酸结合位点的一部分。这个活性位点的位置与诱变研究和mutt样焦磷酸水解酶的序列同源性一致。
The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed beta-sheet connected by the loop I-alpha-helix I-loop II motif, by two tight turns, and by loop III, and terminated by loop IV-alpha-helix II [Abeygunawardana et al. (1993) Biochemistry 32, 13071-13080; Weber et al. (1993) Biochemistry 32, 13081-13087). Complete side-chain assignments of H-1 and C-13 resonances have now been made by 3D C(CO)NH and HCCH-TOCSY experiments. A total of 1461 interproton proximities (11 per residue), obtained by 3D N-15-resolved NOESY-HSQC and 3D C-13-resolved NOESY-HSQC spectra, including 372 long-range NOEs, as well as 65 dihedral angle (phi) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure of MutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globular and compact with the parallel portion of the beta-sheet sandwiched between the two alpha-helices, forming an alpha + beta fold. The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick et al. (1995) Biochemistry 34, 5577-5586]. The tertiary structure of MutT shows these residues to be near each other along the loop I-helix I region of the enzyme. A cluster of five glutamate residues (41, 53, 56, 57, and 98) form a patch of strongly negative electrostatic potential Likely constituting the metal binding site. This site is contiguous with a deep cleft between beta-strands A, C, and D and loop I which may contribute to the nucleotide binding site. This location of the active site is consistent with mutagenesis studies and with sequence homologies among MutT-like pyrophosphohydrolases.