The residue mass of L-pyrrolysine in three distinct methylamine methyltransferases
The residue mass of L-pyrrolysine in three distinct methylamine methyltransferases
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DOI：
10.1074/jbc.m506402200
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发表时间：
2005-11-04
影响因子：
4.8
通讯作者：
Krzycki, JA
中科院分区：
文献类型：
--
作者：
Soares, JA;Zhang, LW;Krzycki, JA
在编码MtmB、MtbB或MttB的基因中发现了单个框内琥珀（UAG）密码子，MtmB、MtbB或MttB是在某些细菌中分别从一甲胺、二甲胺或三甲胺起始甲烷形成的甲基转移酶。MtmB的晶体结构表明琥珀密码子编码吡咯赖氨酸，这是自然界中发现的第22种遗传编码氨基酸。以前试图通过质谱法可视化琥珀编码残基的尝试只确定了赖氨酸，留下关于吡咯赖氨酸的存在和结构的信息完全依赖于单一蛋白质的晶体学。在这里，我们报告成功的质谱表征天然存在的吡咯赖氨酸和琥珀编码的蛋白质中的MtmB以外的残基的第一次演示。通过串联质谱法对来自乙腈变性蛋白质的胰凝乳蛋白酶片段进行测序，揭示了MtmB、MtbB和MttB中琥珀编码残基的质量为237.2 +/- 0.2 Da。傅立叶变换离子回旋共振质谱法对吡咯赖氨酰残基进行了准确测量，为237.1456 Da，在基于经验式C12 H19 N3 O2的预测质量的误差范围内。这些测量结果支持MtmB中吡咯赖氨酸的结构为与赖氨酸的N-酰基酰胺键合的4-甲基吡咯啉-5-羧酸酯，但不支持其中吡咯啉环的4-取代基是胺基的替代结构。在所有三种甲基转移酶中具有统计学上相同质量的吡咯赖氨酸的存在与所提出的在UAG密码子的翻译过程中吡咯赖氨酸直接掺入蛋白质中一致，并且表明MtbB和MttB可以利用吡咯赖氨酸在催化过程中的不寻常的亲电性。
Single in-frame amber (UAG) codons are found in the genes encoding MtmB, MtbB, or MttB, the methyltransferases initiating methane formation from monomethylamine, dimethylamine, or trimethylamine, respectively, in certain Archaea. The crystal structure of MtmB demonstrated that the amber codon codes for pyrrolysine, the 22nd genetically encoded amino acid found in nature. Previous attempts to visualize the amber-encoded residue by mass spectrometry identified only lysine, leaving information on the existence and structure of pyrrolysine resting entirely on crystallography of a single protein. Here we report successful mass spectral characterization of naturally occurring pyrrolysine and the first demonstration of the amber-encoded residue in proteins other than MtmB. The sequencing of chymotryptic fragments from acetonitrile-denatured proteins by tandem mass spectrometry revealed the mass of the amber-encoded residue in MtmB, MtbB, and MttB as 237.2 +/- 0.2 Da. Fourier transform ion cyclotron resonance mass spectrometry produced an accurate measurement for the pyrrolysyl-residue as 237.1456 Da, within error limits of the predicted mass based on the empirical formula C12H19N3O2. These measurements support the structure of pyrrolysine in MtmB as 4-methylpyrroline-5- carboxylate in amide linkage with the N-epsilon of lysine but not the alternative structure in which the 4-substituent of the pyrroline ring is an amine group. The presence of pyrrolysine with statistically identical mass in all three methyltransferases is in keeping with the proposed direct incorporation of pyrrolysine into protein during translation of the UAG codon and suggests that MtbB and MttB may exploit the unusual electrophilicity of pyrrolysine during catalysis.