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

Authors

Abigail R. Green, Washington State University
Kevin M. Lewis, Washington State University
John T. Barr, Washington State University
Jeffrey P. Jones, Washington State UniversityFollow
Fachuang Lu, University of Wisconsin-Madison
John Ralph, University of Wisconsin-MadisonFollow
Wilfred Vermerris, University of FloridaFollow
Scott E. Sattler, USDA-ARSFollow
ChulHee Kang, Washington State UniversityFollow

Document Type

Article

Date of this Version

8-2014

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.

Comments

U.S. government work.

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-cisaldehydes 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-transinterconversion.