Abeygunawardana C, Weber D J, Gittis A G, Frick D N, Lin J, Miller A F, Bessman M J, Mildvan A S
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205-2185, USA.
Biochemistry. 1995 Nov 21;34(46):14997-5005. doi: 10.1021/bi00046a006.
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 1H and 13C 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 15N-resolved NOESY-HSQC and 3D 13C-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].(ABSTRACT TRUNCATED AT 250 WORDS)
MutT酶（129个残基）通过取代极少被攻击的β - P，催化正常和诱变的核苷三磷酸（如8 - 氧代 - dGTP）的水解，生成NMP和焦磷酸。先前对MutT的异核NMR研究表明，其二级结构由一个五链混合β - 折叠组成，通过环I - α - 螺旋I - 环II基序、两个紧密转角以及环III相连，并以环IV - α - 螺旋II结束[阿贝古纳瓦德纳等人（1993年）《生物化学》32卷，13071 - 13080页；韦伯等人（1993年）《生物化学》32卷，13081 - 13087页]。现在通过3D C(CO)NH和HCCH - TOCSY实验完成了1H和13C共振的全侧链归属。通过3D 15N分辨NOESY - HSQC和3D 13C分辨NOESY - HSQC谱获得了总共1461个质子间接近度（每个残基11个），包括372个长程NOE，以及65个二面角（φ）约束和34个主链氢键约束，用于通过距离几何、模拟退火以及使用X - PLOR程序进行能量最小化来确定MutT的三级结构。该结构呈球状且紧凑，β - 折叠的平行部分夹在两个α - 螺旋之间，形成α + β折叠。先前已通过NMR弛豫方法表明，必需的二价阳离子结合在残基Gly - 37、Gly - 38、Lys - 39和Glu - 57附近，并且核苷酸结合在残基Leu - 54和Val - 58附近[弗里克等人（1995年）《生物化学》34卷，5577 - 5586页]。（摘要截断于250字）
