Related Experiment Video
Updated: Jul 13, 2026

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays
Published on: November 29, 2014
Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase
Roberta Magnani1, Nihar R Nayak, Mitra Mazarei
1Department of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, Kentucky 40546-0312, USA.
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.
More Related Videos
Related Concept Videos
Eukaryotic RNA Polymerases
All three eukaryotic RNAPs require specific transcription factors, of which the...
Transfer RNA Synthesis
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
Bacterial RNA Polymerase
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
Conservation of Protein Domains Over Different Proteins
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to form...
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein.
Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...

