The Structure and Catalytic Mechanism of Sorghum bicolor Caffeoyl-CoA O-Methyltransferase

Abstract

Caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase responsible for methylation of the meta-hydroxyl group of caffeoyl-coenzyme A (CoA) on the pathway to monolignols, with their ring methoxylation status characteristic of guaiacyl or syringyl units in lignin. In order to better understand the unique class of type 2 O-methyltransferases from monocots, we have characterized CCoAOMT from sorghum (Sorghum bicolor; SbCCoAOMT), including the SAM binary complex crystal structure and steady-state enzyme kinetics. Key amino acid residues were validated with site-directed mutagenesis. Isothermal titration calorimetry data indicated a sequential binding mechanism for SbCCoAOMT, wherein SAM binds prior to caffeoyl-CoA, and the enzyme showed allosteric behavior with respect to it. 5-Hydroxyferuloyl-CoA was not a substrate for SbCCoAOMT. We propose a catalytic mechanism in which lysine-180 acts as a catalytic base and deprotonates the reactive hydroxyl group of caffeoyl-CoA. This deprotonation is facilitated by the coordination of the reactive hydroxyl group by Ca(2+) in the active site, lowering the pKa of the 3'-OH group. Collectively, these data give a new perspective on the catalytic mechanism of CCoAOMTs and provide a basis for the functional diversity exhibited by type 2 plant OMTs that contain a unique insertion loop (residues 208-231) conferring affinity for phenylpropanoid-CoA thioesters. The structural model of SbCCoAOMT can serve as the basis for protein engineering approaches to enhance the nutritional, agronomic, and industrially relevant properties of sorghum.

Figure 1.

A, Global structure of SbCCoAOMT. SbCCoAOMT forms a dimer and adopts a Rossman-like fold to bind SAM. Each subunit of SbCCoAOMT is depicted with ribbon diagrams in green and magenta, and the insertion loop region is highlighted in yellow in both subunits. The space-filling model of SAM and Ca²⁺ are colored in orange and cyan respectively. Molecular graphics images were produced using the UCSF Chimera package. B, Ca²⁺ coordination observed in SbCCoAOMT. Asp-177, Asp-203, Asn-204, and three water molecules coordinate the active site Ca²⁺ of SbCCoAOMT. The purple grid represents an electron density at sigma level 2 of the 2F_o-F_c map. Molecular graphics images were produced using the UCSF Chimera package. C, SAM-binding region of SbCCoAOMT. SAM is coordinated through a combination of hydrogen bonds and hydrophobic interactions. The blue grid represents an electron density at sigma level 2 of the 2F_o-F_c map. Molecular graphics images were produced using the UCSF Chimera package.

Figure 2.

SbCCoAOMT has two clefts that allow for access to active-site Ca²⁺. The box on the left image shows a location of one cleft near the dimer interface (white dotted line) of SbCCoAOMT that allows for Ca²⁺ access. The box on the right image, which is rotated 45° relative to the left image, displays the other cleft that also allows Ca²⁺ access. SAM and Ca²⁺ are shown as red and green spheres, respectively. Molecular graphics images were produced using the UCSF Chimera package.

Figure 3.

Binding site of caffeoyl-CoA in SbCCoAOMT. The conformation and location for caffeoyl-CoA binding were established by a molecular docking approach. Caffeoyl-CoA is shown in green and orange, SAM in blue, Ca²⁺ in magenta, and CCoAOMT in tan. Molecular graphics images were produced using the UCSF Chimera package.

Figure 4.

Measurements of binding through ITC experiments. A, SbCCoAOMT displayed significant affinity for SAM and SAH but no affinity for either caffeic acid or CoA. Although SbCCoAOMT was pretreated with a 3:1 molar ratio of SAH, caffeic acid and CoA did not show any affinity to SbCCoAOMT. B, Caffeoyl-CoA showed affinity to SbCCoAOMT only in the presence of SAH, but under the same conditions, feruloyl-CoA, 5-hydroxyferuloyl-CoA, and p-coumaroyl-CoA did not. C, K180A mutant SbCCoAOMT displayed similar affinity to SAM as the wild type, but D252A mutant CCoAOMT has significantly reduced affinity.

Figure 5.

SbCCoAOMT demonstrates cooperative kinetic activity for caffeoyl-CoA. Plots show kinetic data of wild-type (WT), K180A, and D252A SbCCoAOMT. The two mutant SbCCoAOMTs have no activity toward caffeoyl-CoA, whereas the wild-type curve reflects distinctive cooperative behavior with a Hill coefficient of 2, a K_half of 4.21 ± 0.134 µm, and a V_max of 259.8 pkat mg⁻¹, indicated by dotted lines.

Figure 6.

Sequence alignment of SbCCoAOMT, MsCCoAOMT, McPFOMT, and related OMTs. Sequences used for the alignment were obtained by BLAST searches using SbCCoAOMT and MsCCoAOMT as query sequences as well as McPFOMT. Above the aligned sequences, α-helices and β-sheets are indicated by coils and arrows, respectively. Fully conserved residues are indicated by black boxes with white letters. Conservative substitutions are shown in black boldface letters, and regions of conservative substitutions are indicated by gray shading. Nonconservative substitutions have no special formatting. Asterisks indicate residues that have been identified as putatively important for catalysis, substrate binding, or substrate specificity.

Figure 7.

Sequence alignment of SbCCoAOMT and the proteins encoded by six sorghum CCoAOMT-like genes. α-Helices and β-sheets are indicated by coils and arrows, respectively. Fully conserved residues are indicated by black boxes with white letters. Conservative substitutions are shown in black boldface letters, and regions of conservative substitutions are indicated by gray shading. Nonconservative substitutions have no special formatting. Asterisks indicate residues that have been identified as putatively important for catalysis, substrate binding, or substrate specificity.

Figure 8.

Homology-based models of the active sites of four putative sorghum CCOAOMTs: SbCCoAOMT (A), Sobic.002G242300 (B), Sobic.007G218500 (C), Sobic.007G218700 (D), and Sobic.007G218800 (E). Side chains of conserved residues involved in Ca²⁺ and SAM binding are shown. The active site Ca²⁺ and SAM are shown as a light green sphere-and-orange stick model. Molecular graphics images were produced using the UCSF Chimera package.

Figure 9.

Proposed catalytic reaction mechanism of SbCCoAOMT. Lys-180 deprotonates the 3ʹ-OH of caffeoyl-CoA, allowing it to nucleophilically attack the methyl group of SAM, forming feruloyl-CoA and SAH.