Burkholderia sp. SG-MS1 and Pseudomonas sp. SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here we use comparative genomics, proteomics, protein semi-purification and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 which carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a Km of 1.17 μM for (+)-pinoresinol. The enzyme is over-expressed in the strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1 kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7 kb cluster in SG-MS1. The amino acid identities of four of the other proteins encoded within the SG-MS2 cluster suggest they could catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome.

IMPORTANCE

(+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β' linkages in lignin. We report a gene cluster in a Pseudomonas and a Burkholderia strain which is involved in oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work throws new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

伯克霍尔德氏菌 SG-MS1和假单胞菌sp。先前已发现SG-MS2通过常见的分解代谢途径使（+）-松脂醇矿化。在这里，我们使用比较基因组学，蛋白质组学，蛋白质半纯化和异源表达，从SG-MS2的香草醇氧化酶/对甲酚甲基羟化酶（VAO / PCMH）酶家族中鉴定出黄素蛋白，该蛋白进行（+ ）-松脂醇在苄基碳上。同源基因与下游细胞色素基因翻译偶联，并且细胞色素是活性所必需的。黄素蛋白具有VAO / PCMH家族辅因子结合和细胞色素需求的独特组合。异源表达的酶具有一个K（+）-松脂醇的m为1.17μM。暴露于（+）-松脂醇后，该酶在SG-MS2菌株中过表达，另外45种其他蛋白质也被发现，其中22种由大约35.1 kb簇中的基因编码，其中也包含黄素蛋白和细胞色素基因。在SG-MS1的38.7 kb簇中也发现了这22个基因中的18个的同系物，以及黄素蛋白和细胞色素基因。SG-MS2簇中编码的其他四个蛋白质的氨基酸身份表明，它们可以催化羟基化的松脂醇转化为原儿茶酸酯和2-甲氧基氢醌。

重要性

（+）-Pinoresinol是重要的植物防御化合物，是人类和其他一些动物的主要食物木脂素，也是用于研究木质素中β-β'键降解的模型化合物。我们报告了假单胞菌和伯克霍尔德尼亚菌株中的基因簇，该菌株参与了（+）-松脂醇的氧化分解代谢。通往该途径的α-羟化酶的黄素蛋白成分属于香草醇氧化酶/ p的4-苯酚氧化（4PO）亚组-甲酚甲基羟化酶（VAO / PCMH）酶家族，但构成该家族辅因子和电子受体性质的新颖组合。它与细胞色素基因翻译偶联，其活性也需要产物。这项工作为（+）-松脂醇的生物学及其向其他生物活性分子的转化提供了新的思路。该发现的潜在应用包括将木质素解构为有用化学物质的新选择，以及从木脂素生成新的植物雌激素性肠内酯。