Research Accomplishment Reports 2007

Mechanism and Significance of Post-Translational Modifications in the Large (LS) and Small (SS) Subunits of Rubisco

R.L. Houtz
Department of Horticulture

 

Project Description

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 (~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  -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.

The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were determined 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)-, where X is any residue, Lys is the methylation site, and  is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including -tocopherol methyltransferase (-TMT). In vitro methylation assays using PsLSMT and a bacterially
expressed form of -TMT from Perilla frutescens confirmed recognition and methylation of -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 -tocopherol, the product of _-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.

            My laboratory continues to characterize the significant decrease in catalytic efficiency of non-methylated fructose-1,6 bisphosphate aldolase from tobacco Rubisco LSMT knock-down plants, and have determined that the loss is due to decreases in both Km and kcat. Currently we are conducting metabolic profile analyses to determine the ramifications of decreased FBP aldolase activity in vivo.

            We also study plant-specific peptide deformylase and the structure of Arabidopsis peptide deformylase 2 (AtDEF2; EC 3.5.1.88), has been determined at a resolution of 2.4 Å. The overall fold and symmetry of DEF2 is similar to eubacterial and mitochondrial forms of DEF (DEF1). DEF2 is essential in plants and has preferred substrate specificity towards the photosystem II D1 polypeptide. A comparative sequence analyses coupled with structural overlays between DEF2, DEF1, and eubacterial DEFs, identified (perhaps we can put here the exact number of residues) several residues which may contribute to the unique catalytic properties of DEF2. Molecular docking models of the N-terminal five amino acids from the D1 polypeptide into the active-site of DEF2 suggest an influence of Tyr-178 in DEF2 as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr-2 in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-termini was performed on DEF2 mutagenized at Tyr-178 to Ala, Phe, or Arg (equivalent residue in DEF1). The results suggest that while Tyr-178 can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

Impact

Understanding the role and structure/function relationships for Rubisco LSMT is uncovering a potentially significant and unknown mechanism for the regulation of several chloroplast-localized enzymes. These findings may be useful for understanding how carbon reactions in the chloroplast can be altered to enhance photosynthetic efficiency. Understanding peptide deformylase provides the unique opportunity to explore potential alternatives to anti-biotic resistance markers in plant transformation vectors as well as alternative broad-spectrum herbicides.

Publications

Magnani, R., N.R. Nayak, M. Mazarei, L.M. Dirk, and R.L. Houtz. 2007. Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase. J. Biol. Chem. 282:27857-27864.

Dirk, L.M., E.M. Flynn, K. Dietzel, J.F. Couture, R.C. Trievel, and R.L. Houtz. 2007. Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases. Biochemistry 46:3905-3915.