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

Journal Article · Mon Jul 25 00:00:00 EDT 2016 · Plant Physiology (Bethesda)

DOI:https://doi.org/10.1104/pp.16.00845· OSTI ID:1768180

Walker, Alexander M. ^[1]; Sattler, Steven A. ^[1]; Regner, Matt  ^[2]; Jones, Jeffrey P. ^[1]; Ralph, John  ^[2]; Vermerris, Wilfred ^[3]; Sattler, Scott E.  ^[4]; Kang, ChulHee  ^[1]

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1. Washington State Univ., Pullman, WA (United States)
2. Univ. of Wisconsin, Madison, WI (United States)
3. Univ. of Florida, Gainesville, FL (United States)
4. US Dept. of Agriculture (USDA), Lincoln, NE (United States)

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. Here, this deprotonation is facilitated by the coordination of the reactive hydroxyl group by Ca²⁺ in the active site, lowering the pK_a 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.

• Research Organization: Univ. of Florida, Gainesville, FL (United States); Univ. of Wisconsin, Madison, WI (United States)
• Sponsoring Organization: USDOE Office of International Affairs (IA); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Transportation Office. Bioenergy Technologies Office; National Science Foundation (NSF); National Institutes of Health (NIH); M.J. Murdock Charitable Trust; USDA
• DOE Contract Number: PI0000031; FC02-07ER64494; MCB 102114; CHE 118359; R01GM11125401; 2011-1006-30358; 2011-67009-30026; 3042-21220-032-00D
• OSTI ID: 1768180
• Journal Information: Plant Physiology (Bethesda), Vol. 172, Issue 1; ISSN 0032-0889
• Publisher: American Society of Plant Biologists
• Country of Publication: United States
• Language: English