Volume 9, Issue 4, Pages (April 2016)

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Volume 9, Issue 4, Pages 569-581 (April 2016) Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants  Sheng Ma, Jacqueline Martin-Laffon, Morgane Mininno, Océane Gigarel, Sabine Brugière, Olivier Bastien, Marianne Tardif, Stéphane Ravanel, Claude Alban  Molecular Plant  Volume 9, Issue 4, Pages 569-581 (April 2016) DOI: 10.1016/j.molp.2016.01.003 Copyright © 2016 The Author Terms and Conditions

Figure 1 Ribbon Representation of the Structure of PsLSMT in Complex with the AdoMet Structural Analog, Aza-adenosyl-L-methionine (AzaAdoMet) (PDB: 2H2E). Enzyme domains constituting the N-terminal and C-terminal lobes are colored differently. AzaAdoMet is displayed as red stick at the cofactor binding site. The figure was produced using PyMOL (DeLano Scientific, San Carlos, CA, USA). Molecular Plant 2016 9, 569-581DOI: (10.1016/j.molp.2016.01.003) Copyright © 2016 The Author Terms and Conditions

Figure 2 Western Blot Analyses of Various Plant Leaf Extracts Using Antibodies Specific to Trimethyl-Lysine. Soluble proteins (40 μg) were resolved by SDS–PAGE and probed with antibodies after electrotransfer onto nitrocellulose membranes (upper panel). Membranes were stained with Ponceau S as controls of protein loading (lower panel). Positions of RBCL, FBAs, and molecular markers are indicated. Molecular Plant 2016 9, 569-581DOI: (10.1016/j.molp.2016.01.003) Copyright © 2016 The Author Terms and Conditions

Figure 3 Identification of an LSMT Region Important for RBCL Methylation Using a Domain-Swapping Strategy. Chimera between AtLSMT and PsLSMT parent enzymes were produced, purified, and assayed for activity with purified recombinant A. thaliana FBA2 (35 μM) or native spinach Rubisco (43 μM on the RBCL subunit basis) as protein substrates. Assays were conducted for 30 min in the presence of 1 μg (parent) or 5 μg (chimeric) LSMTs as described in the Methods section. Proteins were then either precipitated to count methyl groups incorporated into FBA2 and RBCL (central panel) or resolved by SDS–PAGE and used for phosphorimage analysis and Coomassie brilliant blue (CBB) staining (right panel). Values are mean ± SD of three replicates. Positions of RBCL and FBA2 are indicated. Chimeric LSMT proteins are represented by a combination of blue and red boxes indicating the AtLSMT (blue) and PsLSMT (red) peptide regions, respectively (left panel). Positions of the oligonucleotides used to amplify cDNA fragments encoding the LSMT peptide regions are indicated above the chimera representations. The position of exchange sites in chimera is indicated by residue number. Molecular Plant 2016 9, 569-581DOI: (10.1016/j.molp.2016.01.003) Copyright © 2016 The Author Terms and Conditions

Figure 4 Identification by Comparative Sequence Analysis of Amino Acid Residues that Correlates with Difference in LSMT Substrate Specificity. Sequences of the region encompassing the C-terminal portion of the SET domain and the flanking cSET domain from 14 plant LSMTs were aligned using ClustalW. White letters on black background designate conserved residues. Black letters on gray background designate similar residues. Substrate specificity of the aligned LSMTs is indicated on the left. Arrows indicate the position of amino acids specifically differing in correlation with the difference in LSMT substrate specificity (RBCL trimethylation ability). Molecular Plant 2016 9, 569-581DOI: (10.1016/j.molp.2016.01.003) Copyright © 2016 The Author Terms and Conditions

Figure 5 Surface Representation of the Structure of PsLSMT in Complex with AzaAdoMet (PDB: 2H2E). The 88-residue segment of the PsLSMT sequence that plays a crucial role in RBCL methylation is represented in dark gray. Amino acids correlating with the ability of LSMTs to methylate RBCL, on the basis of comparative sequence analysis, are represented in yellow. Blue arrows indicate the predicted orientation of the RBCL peptide substrate within the lysine binding site (Trievel et al., 2003). AzaAdoMet is displayed as red stick at the cofactor binding site. The figure was produced using PyMOL (DeLano Scientific, San Carlos, CA, USA). Molecular Plant 2016 9, 569-581DOI: (10.1016/j.molp.2016.01.003) Copyright © 2016 The Author Terms and Conditions

Figure 6 Influence of Point Mutations on the Substrate Specificity of AtLSMT. Single (A), double (B), and triple (C) AtLSMT mutants were produced, purified, and assayed for activity with purified recombinant A. thaliana FBA2 (35 μM) or native spinach Rubisco (43 μM on the RBCL subunit basis) as protein substrates. Assays were conducted for 30 min in the presence of 1 μg of AtLSMT mutants as described in the Methods section. Proteins were then either precipitated to count methyl groups incorporated into FBA2 and RBCL (upper panels) or resolved by SDS–PAGE and used for phosphorimage analysis and Coomassie brilliant blue (CBB) staining (lower panels). Values are means ± SD of three replicates. Values were corrected for background incorporation into endogenous FBA contaminating purified Rubisco substrate and/or for LSMT automethylation. Positions of RBCL and FBA2 are indicated. Stars indicate AtLSMT mutants displaying some automethylation. Molecular Plant 2016 9, 569-581DOI: (10.1016/j.molp.2016.01.003) Copyright © 2016 The Author Terms and Conditions

Figure 7 Detail of the Structure of the N-Lobe of PsLSMT in Complex with AzaAdoMet and lysine (PDB: 2H2E). The 88-residue segment that plays a crucial role in RBCL methylation is in green. His252, Ala259, and Trp265 residues are displayed as yellow sticks. Bound lysine (Lys) and AzaAdoMet are displayed as blue and red spheres at the substrate and cofactor binding sites, respectively. The figure was produced using PyMOL (DeLano Scientific, San Carlos, CA, USA). Molecular Plant 2016 9, 569-581DOI: (10.1016/j.molp.2016.01.003) Copyright © 2016 The Author Terms and Conditions

Figure 8 Reciprocal Site-Directed Mutagenesis within the β9–β10 Loop Changes the Substrate Specificity of AtLSMT and PsLSMT. AtLSMT (A) and PsLSMT (B) parent and mutant enzymes were produced, purified, and assayed for activity with purified recombinant A. thaliana FBA2 (35 μM) or native spinach Rubisco (43 μM on the RBCL subunit basis) as described in Figure 6. Values are means ± SD of three replicates. Positions of RBCL and FBA2 are indicated. Molecular Plant 2016 9, 569-581DOI: (10.1016/j.molp.2016.01.003) Copyright © 2016 The Author Terms and Conditions

Figure 9 Evolution of LSMTs Substrate Specificity. The phylogenetic analysis of LSMT was restricted to 28 protein sequences identified by BlastP search using AtLSMT as a query (sequence accessions are listed in the legend of Supplemental Figure 1). Selected sequences from the green lineage (green algae, moss, land plants) were aligned using ClustalW, and the phylogenic tree was inferred by using the Maximum Likelihood method based on the Le-Gascuel model (Le and Gascuel, 2008). A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories; +G, parameter = 2.1638). The consensus bootstrap tree is shown and branch support values (in % for 1000 replicates) are indicated. Analyses were done with the Molecular Evolutionary Genetics Analysis software (Mega 6.06) (Tamura et al., 2013). The arrow indicates the shift in LSMT substrate specificity. The sequence of the β9 strand and β9–β10 loop is shown next to the organism name, as inferred by sequence alignment and modeling of LSMT structures using Protein Homology/analogY Recognition Engine V2.0 via the Phyre2 server (www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (Kelley and Sternberg, 2009). Sequences containing the triad motif His-Ala/Pro-Trp are boxed. The predicted substrate specificity of LSMT enzymes is well correlated to available data on RBCL and FBAs trimethylation status in the corresponding organisms ((Houtz et al., 1992; Mininno et al., 2012); this work). +, trimethylated; −, unmethylated; nd, not determined. Molecular Plant 2016 9, 569-581DOI: (10.1016/j.molp.2016.01.003) Copyright © 2016 The Author Terms and Conditions