Publications

• Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase.

  Magnani R., Nayak N.R., Mazarei M., Dirk L.M., Houtz R.L.

  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 constru ... >> More

  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. << Less

  J Biol Chem 282:27857-27864(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Affinity purification of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase.

  Wang P., Royer M., Houtz R.L.

  Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase (Protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the large subunit of Rubisco. In this paper, an affinity purification procedure for pea (Pisum ... >> More

  Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase (Protein methylase III, Rubisco LSMT, EC 2.1.1.43) catalyzes methylation of the epsilon-amino group of Lys-14 in the large subunit of Rubisco. In this paper, an affinity purification procedure for pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT is described and characterized. Spinach (Spinacia oleracea L. cv Melody) Rubisco, a substrate for pea Rubisco LSMT, was immobilized to polyvinylidene fluoride (PVDF) transfer membranes (Immobilon-P) and used as a ligand for the affinity purification of Rubisco LSMT from pea leaf extracts and chloroplast lysates. Pea Rubisco LSMT specifically bound to PVDF-immobilized spinach Rubisco but not to control PVDF membranes which contained immobilized BSA or pea Rubisco. Rubisco LSMT was not eluted by 1 M KCl but was specifically released by S-adenosyl-L-methionine (AdoMet) or spinach Rubisco. Elution of Rubisco LSMT by AdoMet was a result of catalytic methylation of the PVDF-immobilized spinach Rubisco, and was therefore more efficient than elution by the competitive ligand spinach Rubisco. An increase in the specific activity of Rubisco LSMT of approximately 7000-fold was achieved in one step with this affinity purification technique. Rubisco LSMT is a monomeric protein with a molecular mass of approximately 60 kDa. << Less

  Protein Expr Purif 6:528-536(1995) [PubMed] [EuropePMC]

• Characterization of chloroplastic fructose 1,6-bisphosphate aldolases as lysine-methylated proteins in plants.

  Mininno M., Brugiere S., Pautre V., Gilgen A., Ma S., Ferro M., Tardif M., Alban C., Ravanel S.

  In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein refer ... >> More

  In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO(2) through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts. << Less

  J. Biol. Chem. 287:21034-21044(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases.

  Dirk L.M., Flynn E.M., Dietzel K., Couture J.F., Trievel R.C., Houtz R.L.

  Processive versus distributive methyl group transfer was assessed for pea Rubisco large subunit methyltransferase, a SET domain protein lysine methyltransferase catalyzing the formation of trimethyllysine-14 in the large subunit of Rubisco. Catalytically competent complexes between an immobilized ... >> More

  Processive versus distributive methyl group transfer was assessed for pea Rubisco large subunit methyltransferase, a SET domain protein lysine methyltransferase catalyzing the formation of trimethyllysine-14 in the large subunit of Rubisco. Catalytically competent complexes between an immobilized form of des(methyl) Rubisco and Rubisco large subunit methyltransferase were used to demonstrate enzyme release that was co-incident with and dependent on formation of trimethyllysine. Catalytic rate constants determined for formation of trimethyllysine were considerably lower ( approximately 10-fold) than rate constants determined for total radiolabel incorporation from [3H-methyl]-S-adenosylmethionine. Double-reciprocal velocity plots under catalytic conditions favoring monomethyllysine indicated a random or ordered reaction mechanism, while conditions favoring trimethyllysine suggested a hybrid ping-pong mechanism. These results were compared with double-reciprocal velocity plots and product analyses obtained for HsSET7/9 (a monomethyltransferase) and SpCLR4 (a dimethyltransferase) and suggest a predictive ability of double-reciprocal velocity plots for single versus multiple methyl group transfers by SET domain protein lysine methyltransferases. A model is proposed for SET domain protein lysine methyltransferases in which initial binding of polypeptide substrate and S-adenosylmethionine is random, with polypeptide binding followed by deprotonation of the epsilon-amine of the target lysyl residue and subsequent methylation. Following methyl group transfer, S-adenosylhomocysteine and monomethylated polypeptide dissociate from monomethyltransferases, but di- and trimethyltransferases begin a successive and catalytically obligatory deprotonation of enzyme-bound methylated lysyl intermediates, which along with binding and release of S-adenosylmethionine and S-adenosylhomocysteine is manifested as a hybrid ping-pong-like reaction mechanism. << Less

  Biochemistry 46:3905-3915(2007) [PubMed] [EuropePMC]

• Organization and characterization of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase gene in tobacco.

  Ying Z., Janney N., Houtz R.L.

  Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) epsilon N-methyltransferase (Rubisco LSMT) catalyzes the posttranslational methylation of the epsilon-amino group of Lys-14 in the LS of Rubisco in many higher plant species including tobacco. The tobacco Rubisco LSMT gen ... >> More

  Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) epsilon N-methyltransferase (Rubisco LSMT) catalyzes the posttranslational methylation of the epsilon-amino group of Lys-14 in the LS of Rubisco in many higher plant species including tobacco. The tobacco Rubisco LSMT gene (rbcMT-T) and its cDNA were isolated, sequenced, and characterized. The gene contains 6 exons and spans about 6 kb. Primer extension analysis indicated one transcription start site located 93 nt upstream of the translation initiation site. Sequence analysis of the 5'-flanking region suggests several potential binding sites for transcription factors, including 7 GT-1 elements and an HSP-70.5 element. Gene dosage analysis by Southern hybridization demonstrated that the tobacco rbcMT-T gene is present as a single copy in the tobacco haploid genome. The full-length cDNA for tobacco rbcMT-T is 1974 nt in length excluding the 3' poly(A)15 tail, and encodes a 491 amino acid polypeptide with a molecular mass of ca. 56kDa. The deduced amino acid sequence of tobacco Rubisco LSMT has 64.5% identity and 75.3% similarity with the sequence of pea Rubisco LSMT, and both proteins contain several copies of a conserved imperfect leucine-rich repeat motif. << Less

  Plant Mol. Biol. 32:663-671(1996) [PubMed] [EuropePMC]