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. 2014 Sep 12;289(37):25690-8.
doi: 10.1074/jbc.M114.584920. Epub 2014 Jul 30.

Structural insights into the catalytic mechanism of Synechocystis magnesium protoporphyrin IX O-methyltransferase (ChlM)

Xuemin Chen  1 , Xiao Wang  1 , Juan Feng  1 , Yuhong Chen  2 , Ying Fang  1 , Shun Zhao  1 , Aiguo Zhao  1 , Min Zhang  3 , Lin Liu  4
Affiliations

Structural insights into the catalytic mechanism of Synechocystis magnesium protoporphyrin IX O-methyltransferase (ChlM)

Xuemin Chen et al. J Biol Chem. .

Abstract

Magnesium protoporphyrin IX O-methyltransferase (ChlM) catalyzes transfer of the methyl group from S-adenosylmethionine to the carboxyl group of the C13 propionate side chain of magnesium protoporphyrin IX. This reaction is the second committed step in chlorophyll biosynthesis from protoporphyrin IX. Here we report the crystal structures of ChlM from the cyanobacterium Synechocystis sp. PCC 6803 in complex with S-adenosylmethionine and S-adenosylhomocysteine at resolutions of 1.6 and 1.7 Å, respectively. The structures illustrate the molecular basis for cofactor and substrate binding and suggest that conformational changes of the two "arm" regions may modulate binding and release of substrates/products to and from the active site. Tyr-28 and His-139 were identified to play essential roles for methyl transfer reaction but are not indispensable for cofactor/substrate binding. Based on these structural and functional findings, a catalytic model is proposed.

Keywords: Chlorophyll; Chloroplast; Enzyme Mechanism; Enzyme Structure; Photosynthesis; Protein Structure.

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Figures

FIGURE 1.
FIGURE 1.
Overall structure of SyChlM. A, topology diagram of SynChlM. Helices and β-strands are represented by cylinders and arrows. The starting and ending residue numbers for each secondary structural element are labeled. B, ribbon representation of the monomeric SyChlM-SAM. The SAM cofactor is in stick model. The dotted lines indicate the missing sequences. The starting and ending residue numbers for sequences not observed in the electron density are labeled. C, ribbon representation of the two SyChlM-SAH monomers with SAH shown in stick model. D, superimposition of the monomeric forms of SyChlM (as in B and C). A dashed line is drawn between the αG region and the core of SyChlM. The radius of the tubes corresponds to the B factor.
FIGURE 2.
FIGURE 2.
Multiple ChlM sequences alignment and the cofactor binding pocket of SyChlM. A, multiple sequence alignment of ChlMs. Identical amino acids are in white on a red background, with the DXGCGXG motifs on a cyan background. The similar residues are in red and boxed. Dots indicate gaps introduced during alignment. Pink ellipses and green stars denote the residues involved in binding to the cofactor and to MgP, respectively. The two catalytically essential residues are marked with blue triangles. B, electron density maps of SAM and SAH. The 2Fo − Fc maps at 0.5 σ for SAM (top) and SAH (bottom) are shown. C, clipped view of the SAM binding pocket. SyChlM is in gray. D, residues interacting with SAM in the pocket. Residues involved in hydrophobic interaction with SAM are in green; residues involved in hydrogen bond formation are in yellow. The backbone of the DXGCGXG motif is in blue. Two waters forming hydrogen bonds with SAM are shown as red spheres. Hydrogen bonds are depicted as dotted dashes.
FIGURE 3.
FIGURE 3.
Model of SyChlM-SAM-MgP. A, MgP docked into SyChlM. SyChlM is presented in gray, and MgP is in indigo with the magnesium colored green. B, the substrate binding site. Residues predicted to interact with MgP are in green with Tyr-28 and His-139 colored in yellow. Putative hydrogen bonds are depicted as dotted dashes.
FIGURE 4.
FIGURE 4.
Spectrophotometric methyltransferase assay. The reaction mixture was measured by recording absorbance at 265 nm for 5 min. Data are presented as the mean ± S.E. of at least three independent experiments.
FIGURE 5.
FIGURE 5.
ITC analysis of ligands binding. A, titration of SyChlMWT with MgP. The top panel shows the heat response to injections, and the bottom panel shows the integrated heats of each injection (■) and the fit (–) to a single-site binding model. B, titration of SyChlMWT with SAH. Top and bottom panels are the same as in A.
FIGURE 6.
FIGURE 6.
Comparison of SyChlM and its structural homologs. A, backbone superimposition of SyChlM-SAH (gray) and its structural homologs (red to blue) with a Z score above 18. Cooler color corresponds to lower Z score listed in Table 3. B, ribbon superimposition of SyChlM (gray) with DhpI (orange). A dashed line is drawn between the insertion regions and the protein cores. The coordinates of DhpI are from PDB entry 3OU6 (44) to show the capping helix that is not observed in 3OU2.
FIGURE 7.
FIGURE 7.
Model of ChlM catalysis. A, predicted active site models of SyChlM and RhBchM. The homology model of RhBchM was built by SWISS-MODEL using the SyChlM coordinates as template. B, surface model of SyChlM (gray) with the two flexible regions shown as yellow ribbons. Before substrate binding, the N-terminal arm and the αG arm are intrinsically flexible (shown in transparent mode). Upon SAM and MgP binding, these two arms become stable. The right panel shows surface presentation of the whole protein, with these two arms in yellow to illustrate their independent status.
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