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

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.
Resources in this dataset:Resource Title: Crystal Structure of sorghum caffeoyl-CoA O-methyltransferase (CCoAOMT) - Protein Data Bank 5KVA. File Name: Web Page, url: https://www.rcsb.org/structure/5KVA Includes: Structural Summary; 3D View; Macromolecule Annotations; Sequence Display; Sequence Similarity Clusters; Structure Similarities; and X-RAY DIFFRACTION Experimental Data and Validation.

咖啡酰辅酶A 3-O-甲基转移酶（CCoAOMT）是一种依赖S-腺苷甲硫氨酸（SAM）的O-甲基转移酶，负责在单宁醇生物合成途径中甲基化咖啡酰辅酶A（CoA）的亚甲基羟基，其环状甲氧基化状态是木质素中愈创木基或松柏木基单元的特征。为了更深入地理解单子叶植物中独特的第二类O-甲基转移酶类，我们对高粱（Sorghum bicolor；SbCCoAOMT）中的CCoAOMT进行了表征，包括SAM二聚体复合物的晶体结构和稳态酶动力学。通过定点突变验证了关键氨基酸残基。等温滴定 calorimetry数据表明SbCCoAOMT具有连续结合机制，其中SAM在咖啡酰-CoA之前结合，并且该酶在SAM上表现出变构行为。5-羟基阿魏酰-CoA不是SbCCoAOMT的底物。我们提出了一种催化机制，其中赖氨酸-180作为催化碱，去质子化咖啡酰-CoA的活性羟基。这种去质子化过程得益于活性位点中Ca(2+)对反应性羟基的配位作用，降低了3'-OH基团的pKa值。综上所述，这些数据为CCoAOMTs的催化机制提供了新的视角，并为具有独特插入环（残基208-231）的类型2植物OMTs的功能多样性提供了基础，这种插入环赋予其与苯丙素-CoA硫酯的亲和力。SbCCoAOMT的结构模型可作为蛋白质工程方法的基础，以增强高粱的营养、农学和工业相关特性。该数据集中的资源包括：
资源标题：高粱咖啡酰-CoA O-甲基转移酶（CCoAOMT）的晶体结构 - 蛋白质数据银行5KVA。文件名：网页，网址：https://www.rcsb.org/structure/5KVA
包含内容：结构摘要；3D视图；大分子注释；序列显示；序列相似性聚类；结构相似性；以及X-RAY DIFFRACTION实验数据和验证。