Location: Wheat, Sorghum and Forage ResearchTitle: Determination of the structure and catalytic mechanism of Sorghum bicolor caffeic acid O-methyltransferase and the structural impact of three brown midrib12 mutations Author
|Kang, Chul Hee|
Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/17/2014
Publication Date: 8/4/2014
Publication URL: http://handle.nal.usda.gov/10113/59513
Citation: Green, A.R., Lewis, K.M., Barr, J.T., Jones, J.P., Lu, F., Ralph, J., Vermerris, W., Sattler, S.E., Kang, C. 2014. Determination of the structure and catalytic mechanism of Sorghum bicolor caffeic acid O-methyltransferase and the structural impact of three brown midrib12 mutations. Plant Physiology. 165(4):1440-1456. DOI: 10.1104/pp114.241729. Interpretive Summary: In the U.S., sorghum biomass (stalks and leaves) serves as an important forage crop for livestock. In addition, sorghum is being developed as a renewable bioenergy crop for liquid biofuels. There are three main components of biomass: cellulose, hemicellulose and lignin. Lignin impedes conversion of biomass to both energy for livestock and fuel for bioenergy. Therefore, understanding lignin synthesis is critical for developing plants that are improved for bioenergy conversion. The sorghum caffeic O-methyltransferase (COMT) is a key enzyme in lignin synthesis. Mutations in the gene encoding this enzyme result in biomass that has reduced lignin content and improved digestibility for livestock and conversion to biofuels. We determined and examined the structure of this enzyme in order to understand its function in lignin synthesis. The structure of COMT was similar to enzymes found in other plants such as ryegrass and alfalfa. Our observations explain how the sorghum COMT and similar enzymes participate in lignin synthesis, and why COMT is only able to participate in one specific chemical reaction in this pathway. This information will provide new means to alter lignin content in sorghum and other plants, thereby leading to more efficient substrates for bioenergy and livestock conversion.
Technical Abstract: With S-adenosylmethionine (SAM) acting as the methyl donor, caffeic acid O-methyltransferase from Sorghum bicolor (SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde, to form sinapaldehyde. In order to determine the mechanism of SbCOMT and understand the reduction in activity observed in three brown midrib12 (bmr12) mutants that carry missense mutations in the COMT gene and that cause a significant reduction in the syringyl-to-guaiacyl (S/G) ratio in the lignin, we have determined the crystal structures of both the apo-form and the SAM binary complex SbCOMT, and established the ternary complex structure by molecular modeling. These structures have revealed many shared features with the COMTs from both the monocot ryegrass (Lolium perenne) and the dicot alfalfa (Medicago sativa). Steady-state kinetic and isothermal titration calorimetric data reveal that the phenylpropanoid and SAM substrates of SbCOMT enter the enzyme in arbitrary order before the products are released, suggesting a random bi-bi mechanism. Based on the structural, kinetic and thermodynamic results, we propose that the observed hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol and acid substrates is due to the ability of the aldehyde substrate to stabilize, through electron delocalization, the anionic intermediate that results from deprotonation of the hydroxyl group ortho to the 4-OH by His267. The preferential formation of sinapaldehyde as a precursor for S-subunits in vivo, despite the presence of other phenylpropanoids for which SbCOMT has affinity, is a consequence of the narrow range in activity of SbCOMT towards 5-hydroxyconiferaldehyde and the effective substrate inhibition of this compound. We propose that this substrate inhibition is due to the high rotational barrier around the bond connecting the vinyl and aldehyde portions of the substrate with the s-cis-form of these aldehydes having the capability to bind non-productively to the active site and inhibit productive binding of the s-trans-form.