Biology Department, Brookhaven National Laboratory, Upton, NY, United States.
Biology Department, Brookhaven National Laboratory, Upton, NY, United States.
Methods Enzymol. 2022;676:133-158. doi: 10.1016/bs.mie.2022.07.027. Epub 2022 Aug 28.
Lignin is a complex heterogenous polymer derived from oxidative radical polymerization of three monolignols, i.e., p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These lignin monomeric precursors structurally differ in their methoxy groups of the benzene rings. In phenylpropanoid-monolignol biosynthetic pathway, the endoplasmic reticulum (ER)-resident cytochrome P450 monooxygenases, cinnamate 4-hydroxylase, coumaroyl ester 3'-hydroxylase and ferulate 5-hydroxylase, establish the key structural characteristics of monolignols. The catalysis of cytochrome P450 monooxygenase requires reducing power, which is supplied by the ER electron transfer chains, composed of cytochrome P450 oxidoreductase (CPR), cytochrome b reductase (CBR) and/or cytochrome b protein (CB5), from cofactor NADPH or NADH. While NADPH-dependent CPR serves as the typical electron donor for most P450 enzymes, in some cases, the CBR-CB5 or CPR-CB5 electron transfer system also transfers electrons to the terminal P450 enzymes. There are tremendous studies focusing on the discovery and characterization of cytochrome P450 monooxygenases. However, very limited attention has been paid to the versatility and the roles of electron transfer components in the P450 catalytic system. Due to the membrane-residence property of both P450 enzymes and electron transfer components, it is challenging to establish an effective experimental system to evaluate the functional association of P450s with their redox partners. This chapter describes a yeast cell biocatalytic system and the related experimental procedures for comparatively assessing the functional relationship of monolignol biosynthetic P450 enzymes and different redox partners in their catalysis.
木质素是一种复杂的杂聚物，由三种单体酚，即对香豆醇、松柏醇和芥子醇，通过氧化自由基聚合而成。这些木质素单体前体在苯环的甲氧基结构上有所不同。在苯丙烷单体酚生物合成途径中，内质网（ER）驻留细胞色素 P450 单加氧酶，肉桂酸 4-羟化酶、松柏酰基酯 3'-羟化酶和阿魏酸 5-羟化酶，为单体酚建立了关键的结构特征。细胞色素 P450 单加氧酶的催化需要还原力，这由 ER 电子传递链提供，由细胞色素 P450 氧化还原酶（CPR）、细胞色素 b 还原酶（CBR）和/或细胞色素 b 蛋白（CB5）组成，来自辅因子 NADPH 或 NADH。虽然 NADPH 依赖的 CPR 作为大多数 P450 酶的典型电子供体，但在某些情况下，CBR-CB5 或 CPR-CB5 电子传递系统也将电子转移到末端 P450 酶。有大量研究集中在细胞色素 P450 单加氧酶的发现和特性上。然而，对于电子传递成分在 P450 催化系统中的多功能性和作用，关注非常有限。由于 P450 酶和电子传递成分都具有膜保留性质，因此建立一个有效的实验系统来评估 P450 与它们的氧化还原伴侣的功能关联具有挑战性。本章描述了一个酵母细胞生物催化系统和相关的实验程序，用于比较评估单体酚生物合成 P450 酶与不同氧化还原伴侣在其催化中的功能关系。
