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J. Biol. Chem., Vol. 280, Issue 44, 36962-36969, November 4, 2005

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The Residue Mass of L-Pyrrolysine in Three Distinct Methylamine Methyltransferases^*

Jitesh A. Soares, Liwen Zhang, Rhonda L. Pitsch, Nanette M. Kleinholz, R. Benjamin Jones, Jeremy J. Wolff¶, Jon Amster¶1, Kari B. Green-Church2, and Joseph A. Krzycki||3

From the Department of Microbiology, Campus Chemical Instrument Center Mass Spectrometry and Proteomics Facility, and the ^||Ohio State University Biochemistry Program, Ohio State University, Columbus, Ohio 43210 and the ^¶Department of Chemistry, University of Georgia, Athens, Georgia 30602

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 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.

Received for publication, June 13, 2005 , and in revised form, July 28, 2005.

^* Purchase of the mass spectrometry equipment was supported by the Hayes Academic Investment Funds of the Ohio Board of Regents. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S4.

¹ Supported by National Science Foundation Grant CHE-0316002.

² To whom correspondence may be addressed: Mass Spectrometry and Proteomics Facility, 243 Fontana Labs, 116 W. 19th Ave., Columbus OH 43210. Tel.: 614-688-0521; Fax: 614-292-5955; E-mail: Green-Church.1{at}osu.edu.

³ Supported by Department of Energy Grant DE-FG0202-91ER200042 and National Science Foundation Grant MCB-9808914. To whom correspondence may be addressed: Dept. of Microbiology, Ohio State University, 484 West 12th Ave., Columbus, OH 43210. Tel.: 614-292-1578; Fax: 614-292-8120; E-mail: Krzycki.1{at}osu.edu.

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