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The Structure and Catalytic Mechanism of Sorghum bicolor Caffeoyl-CoA O-Methyltransferase1

The catalytic mechanism and substrate specificity of caffeoyl-CoA O-methyltransferase from Sorghum bicolor deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Caffeoyl-coenzyme A 3- O -methyltransferase (CCoAOMT) is an S -adenosyl methionine ( SAM )-de...

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Published in:Plant physiology (Bethesda) 2016-07, Vol.172 (1), p.78-92
Main Authors: Walker, Alexander M., Sattler, Steven A., Regner, Matt, Jones, Jeffrey P., Ralph, John, Vermerris, Wilfred, Sattler, Scott E., Kang, ChulHee
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container_issue 1
container_start_page 78
container_title Plant physiology (Bethesda)
container_volume 172
creator Walker, Alexander M.
Sattler, Steven A.
Regner, Matt
Jones, Jeffrey P.
Ralph, John
Vermerris, Wilfred
Sattler, Scott E.
Kang, ChulHee
description The catalytic mechanism and substrate specificity of caffeoyl-CoA O-methyltransferase from Sorghum bicolor deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. 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 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.
doi_str_mv 10.1104/pp.16.00845
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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 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. 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title The Structure and Catalytic Mechanism of Sorghum bicolor Caffeoyl-CoA O-Methyltransferase1
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