Polypeptide substrate specificity of PsLSMT: A set domain protein methyltransferase

Roberta Magnani, Nihar R. Nayak, Mitra Mazarei, Lynnette M.A. Dirk, Robert L. Houtz

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9 Scopus citations


Rubisco large subunit methyltransferase (PsLSMT) is a SET domain protein responsible for the trimethylation of Lys-14 in the large subunit of Rubisco. The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were investigated using a fusion protein construct between the first 23 amino acids from the large subunit of Rubisco and human carbonic anhydrase II. A total of 40 conservative and non-conservative amino acid substitutions flanking the target Lys-14 methylation site (positions P -3 to P+3) were engineered in the fusion protein. The catalytic efficiency (kcat/Km) of PsLSMT was determined using each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-π, where X is any residue, Lys is the methylation site, and π is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including γ-tocopherol methyltransferase (γ-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of γ-TMT from Perilla frutescens confirmed recognition and methylation of γ-TMT by PsLSMT in vitro. RNA interference-mediated knockdown of the PsLSMT homologue (NtLSMT) in transgenic tobacco plants resulted in a 2-fold decrease of α-tocopherol, the product of γ-TMT. The results demonstrate the efficacy of consensus sequence-driven identification of alternative substrates for PsLSMT as well as identification of functional attributes of protein methylation catalyzed by LSMT.

Original languageEnglish
Pages (from-to)27857-27864
Number of pages8
JournalJournal of Biological Chemistry
Issue number38
StatePublished - Sep 21 2007

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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