Rhonda Pitsch - The Residue Mass of L-Pyrrolysine in Three Distinct Methylamine Methyltransferases

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      Publication Details (including relevant citation   information):

      J. Biol. Chem., 2005; 280, 36962-36969.


      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   C(12)H(19)N(3)O(2). These measurements support the structure of   pyrrolysine in MtmB as 4-methylpyrroline-5-carboxylate in amide   linkage with the (epsilon)N 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.

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