title: The Structure and Catalytic Mechanism of Sorghum bicolor Caffeoyl-CoA O-Methyltransferase.
abstract: 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 pKa 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.
authors.affiliation: School of Molecular Biosciences (A.M.W., S.A.S., C.K.) and Department of Chemistry (J.P.J., C.K.), Washington State University, Pullman, Washington 99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53726 (M.R., J.R.);Department of Microbiology and Cell Science and Genetics Institute, University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States Department of Agriculture-Agricultural Research Service, Grain Forage and Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.).
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Walker
authors.affiliation: School of Molecular Biosciences (A.M.W., S.A.S., C.K.) and Department of Chemistry (J.P.J., C.K.), Washington State University, Pullman, Washington 99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53726 (M.R., J.R.);Department of Microbiology and Cell Science and Genetics Institute, University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States Department of Agriculture-Agricultural Research Service, Grain Forage and Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.).
authors.forename: Steven A
authors.initials: SA
authors.lastname: Sattler
authors.affiliation: School of Molecular Biosciences (A.M.W., S.A.S., C.K.) and Department of Chemistry (J.P.J., C.K.), Washington State University, Pullman, Washington 99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53726 (M.R., J.R.);Department of Microbiology and Cell Science and Genetics Institute, University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States Department of Agriculture-Agricultural Research Service, Grain Forage and Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.).
authors.forename: Matt
authors.identifier: 0000-0001-7799-2704
authors.initials: M
authors.lastname: Regner
authors.affiliation: School of Molecular Biosciences (A.M.W., S.A.S., C.K.) and Department of Chemistry (J.P.J., C.K.), Washington State University, Pullman, Washington 99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53726 (M.R., J.R.);Department of Microbiology and Cell Science and Genetics Institute, University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States Department of Agriculture-Agricultural Research Service, Grain Forage and Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.).
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Jones
authors.affiliation: School of Molecular Biosciences (A.M.W., S.A.S., C.K.) and Department of Chemistry (J.P.J., C.K.), Washington State University, Pullman, Washington 99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53726 (M.R., J.R.);Department of Microbiology and Cell Science and Genetics Institute, University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States Department of Agriculture-Agricultural Research Service, Grain Forage and Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.).
authors.forename: John
authors.identifier: 0000-0002-6093-4521
authors.initials: J
authors.lastname: Ralph
authors.affiliation: School of Molecular Biosciences (A.M.W., S.A.S., C.K.) and Department of Chemistry (J.P.J., C.K.), Washington State University, Pullman, Washington 99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53726 (M.R., J.R.);Department of Microbiology and Cell Science and Genetics Institute, University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States Department of Agriculture-Agricultural Research Service, Grain Forage and Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.).
authors.forename: Wilfred
authors.initials: W
authors.lastname: Vermerris
authors.affiliation: School of Molecular Biosciences (A.M.W., S.A.S., C.K.) and Department of Chemistry (J.P.J., C.K.), Washington State University, Pullman, Washington 99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53726 (M.R., J.R.);Department of Microbiology and Cell Science and Genetics Institute, University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States Department of Agriculture-Agricultural Research Service, Grain Forage and Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.).
authors.forename: Scott E
authors.identifier: 0000-0002-6814-4073
authors.initials: SE
authors.lastname: Sattler
authors.affiliation: School of Molecular Biosciences (A.M.W., S.A.S., C.K.) and Department of Chemistry (J.P.J., C.K.), Washington State University, Pullman, Washington 99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, Wisconsin 53726 (M.R., J.R.);Department of Microbiology and Cell Science and Genetics Institute, University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States Department of Agriculture-Agricultural Research Service, Grain Forage and Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.) chkang@wsu.edu.
authors.forename: ChulHee
authors.identifier: 0000-0002-0693-7860
authors.initials: C
authors.lastname: Kang
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D045868:Sorghum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D000214:Acyl Coenzyme A; D010940:Plant Proteins; C058645:caffeoyl-coenzyme A; D008780:Methyltransferases; C059825:caffeoyl-CoA O-methyltransferase; D008239:Lysine; D002118:Calcium