title: Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase.
abstract: 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 (k(cat)/K(m)) 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)-pi, where X is any residue, Lys is the methylation site, and pi is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including gamma-tocopherol methyltransferase (gamma-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of gamma-TMT from Perilla frutescens confirmed recognition and methylation of gamma-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 alpha-tocopherol, the product of gamma-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.
authors.affiliation: Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, Kentucky 40546-0312, USA.
authors.forename: Roberta
authors.initials: R
authors.lastname: Magnani
authors.forename: Nihar R
authors.initials: NR
authors.lastname: Nayak
authors.forename: Mitra
authors.initials: M
authors.lastname: Mazarei
authors.forename: Lynnette M A
authors.initials: LM
authors.lastname: Dirk
authors.forename: Robert L
authors.initials: RL
authors.lastname: Houtz
mesh_terms: D002384:Catalysis; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D031344:Perilla frutescens / Q000201:enzymology* / Q000378:metabolism; D011496:Protein Methyltransferases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics; D024502:alpha-Tocopherol / Q000378:metabolism
chemical_list: D010455:Peptides; D008780:Methyltransferases; D011496:Protein Methyltransferases; C097344:ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit epsilonN-methyltransferase; D011495:Histone-Lysine N-Methyltransferase; C047259:gamma-tocopherol methyltransferase; D012273:Ribulose-Bisphosphate Carboxylase; D024502:alpha-Tocopherol; D008239:Lysine