Determination of the Structure and Catalytic Mechanism of Sorghum bicolor Caffeic Acid O-Methyltransferase and the Structural Impact of Three brown midrib12 Mutations

Abstract

Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol, and acid substrates arises from the ability of the aldehyde to stabilize the anionic intermediate that results from deprotonation of the 5-hydroxyl group by histidine-267. Additionally, despite the presence of other phenylpropanoid substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by its low K_m for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates, the aldehydes exhibit product inhibition, and we propose that this is due to nonproductive binding of the S-cis-form of the aldehydes inhibiting productive binding of the S-trans-form. The S-cis-aldehydes most likely act only as inhibitors, because the high rotational energy barrier around the 2-propenyl bond prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond rotational energy barrier enables rapid S-cis/S-trans-interconversion.

Figure 1.

Molecular mass determination of SbCOMT. MALLS was performed using a 2 mg mL⁻¹ SbCOMT solution. The chromatogram is shown as A₂₈₀ (left y axis) and mass in kD (logarithmic; right y axis) versus elution volume (mL). The dots in the middle of the peak indicate the molecular mass (approximately 80 kD) calculated from the light scattering, indicating the dimeric nature of SbCOMT. A.U., Absorbance units.

Figure 2.

Domain shifts of SbCOMT. A, Cα least-squares superposition of apo-SbCOMT (blue), its SAM complex (green), and LpCOMT (yellow) to illustrate bound substrate-induced domain shifts. SAM in the SbCOMT active site is shown as light-gray spheres for carbons, blue spheres for nitrogen atoms, and red spheres for oxygen atoms; the coloration for SAH and sinapaldehyde in the LpCOMT active site are the same except that dark-gray spheres represent carbons. B, Closeup of the SbCOMT active site with bound SAM. Amino acid residues are labeled, and hydrogen bonds between residue side chains and SAM are shown as black dashed lines. These images were generated using the PyMOL Molecular Graphics System, version 1.3 (Schrödinger).

Figure 3.

Multiple sequence alignment of COMTs from sorghum (SbCOMT), ryegrass (LpCOMT), alfalfa (MsCOMT), and C. breweri (CbCOMT). The α-helices and β-strands of SbCOMT are depicted with blue tubes and pink arrows, respectively. The SAM- and phenylpropanoid-binding residues are indicated by yellow and green highlights, respectively, and the identical residues among the compared sequences are in boldface. Asterisks indicate the locations of point mutations observed in sorghum bmr12 mutants. Multiple sequence alignment was performed with ClustalW using a BLOSUM weighting matrix.

Figure 4.

Ribbon diagram representing the crystal structure of SbCOMT with bound SAM. The SbCOMT dimer is displayed as a ribbon diagram with bound SAM (orange spheres). The positions of the point mutations in the bmr12 mutants are shown as light-blue spheres. This image was generated using the PyMOL Molecular Graphics System, version 1.3 (Schrödinger).

Figure 5.

Measurement of SbCOMT substrate binding through ITC experiments. A and B, Trend of heat released by serial injections of SAM (squares) and SAH (circles) at pH 6.5 (A) and pH 7.5 (B). C, Trend of heat released by serial injections of caffeoyl CoA (circles), cinnamic acid (triangles), and caffeic acid (squares). D, Trend of heat released by serial injections of cinnamaldehyde (diamonds), p-coumaraldehyde (squares), coniferaldehyde (circles), and 5-hydroxyconiferaldehyde (triangles). Solid lines represent the least-square fits of the data.

Figure 6.

Kinetic activity of SbCOMT. A, Kinetic curve of SbCOMT reacting with varying concentrations of 5-hydroxyconiferaldehyde at pH 6.5 (squares) and pH 7.5 (triangles). B and C, Kinetic curves of SbCOMT with varying concentrations of caffeic acid (B) or SAM (C).

Figure 7.

Sequential binding mechanism of SbCOMT. A and B, Lineweaver-Burk plots of SbCOMT activity at various concentrations of caffeic acid with respect to SAM (A) and at various concentrations of SAM with respect to caffeic acid (B). C and D, Lineweaver-Burk plots of SbCOMT activity at varying SAH inhibitory concentrations with respect to SAM (C) and caffeic acid (D). E and F, Lineweaver-Burk plots of SbCOMT activity at varying ferulic acid inhibitory concentrations with respect to SAM (E) and caffeic acid (F). G, Cleland notation of the predicted random bi-bi mechanism of SbCOMT. CA, Caffeic acid; FA, ferulic acid.

Figure 8.

CD spectra for SbCOMT from wild-type sorghum (WT) and bmr12 mutants. The 200- to 300-nm CD spectra of the wild-type SbCOMT and its four bmr12 mutants were compared in the same condition (5 µm in phosphate-buffered saline) using an AVIV 202SF spectropolarimeter (AVIV Biomedical) at 25°C. The similar shapes and intensities for the far UV light-CD spectra indicate that wild-type SbCOMT, the A71V mutant, and the G325S mutant contain similar amounts of secondary structure, while the P150L mutant and the A71V, P150L double mutant have reduced secondary structure.

Figure 9.

SbCOMT active-site electrostatic potential surface with modeled 5-hydroxyconiferaldehyde and hydrogen bonding interactions. A and B, In A (with 5-hydroxyconiferaldehyde) and B (apo), the modeled active-site amides (Asn-128 and Asn-323) are present as propanamide, Asp-268 is present as propanoate, Glu-296 and Glu-328 are present as acetate, Met-317 is present as ethyl methyl sulfide, and SAM is present as diethylmethylsulfonium. The active site is mapped at an iso value of 0.020 electrons Bohr⁻³ and shown on a potential scale of −3.10 × 10¹ Hartrees (red) to +4.00 Hartrees (blue). These images were generated using GaussView 3.09. C, The modeled active site hydrogen bonding interactions. Hydrogen bonds are shown as black dotted lines. The green cartoon represents the protein forming the active site, and the yellow α-helix corresponds to the dimeric partner’s α1. This image was generated using the PyMOL Molecular Graphics System, version 1.3 (Schrödinger).

Figure 10.

Catalytic mechanism and productive versus nonproductive substrate binding. A, I, 5-Hydroxyconiferaldehyde and SAM bind to the active site with a concomitant release of solvent and active-site closure. Asp-268 aids in reorienting the 4- and 5-hydroxyl groups from the syn-conformation favored by the phenylpropanoid molecule to the anti-like configuration required for catalysis. II, His-267 abstracts a proton from the 4-hydroxyl group, which is then methylated by SAM. III, SAH and sinapaldehyde product complex prior to COMT reopening. IV, SAH and sinapaldehyde leave, and the basic form of His-267 is regenerated by losing its acidic proton to solvent. B, Productive binding by S-trans-5-hydroxyconiferaldehyde (left) and nonproductive binding by S-cis-5-hydroxyconiferaldehyde. The positions of the 3-methoxyl and 5-hydroxyl groups of S-cis-5-hydroxyconiferaldehyde are switched in the binding site so that the methoxy group is oriented toward His-267 and SAM.

Figure 11.

A 360° 5-hydroxyconiferyl substrate φ_7-8-9-O dihedral angle potential energy surface. The φ_7-8-9-O dihedral angle potential energy surface scans are from 5-hydroxyconiferaldehyde (solid lines), 5-hydroxyconiferyl alcohol (dashed lines), and 5-hydroxyferulate (dotted lines) with gas-phase energies for each molecule set relative to their lowest-energy φ_7-8-9-O dihedral angle of 180° for 5-hydroxyconiferaldehyde, 119.94° for 5-hydroxyconiferyl alcohol, and 180° for 5-hydroxyferulate. The 30° incremental scan points for 5-hydroxyconiferaldehyde (circles), 5-hydroxyconiferyl alcohol (squares), and 5-hydroxyferulate (triangles) are shown.