A variable loop involved in the substrate selectivity of pinoresinol/lariciresinol reductase from Camellia sinensis. (June 2019)

• Record Type: Journal Article
• Authors: Wu, Yingling; Xing, Dawei; Ma, Guoliang; Dai, Xinlong; Gao, Liping; Xia, Tao
• Is Part Of: Phytochemistry. Volume 162(2019)
• Page Start: 1
• Page End: 9
• Publication Date: 2019-06
• Subjects: Camellia sinensis var. sinensis cv. Shuchazao -- Pinoresinol/lariciresinol reductase -- Variable loop -- Substrate selectivity; CsPLR pinoresinol/lariciresinol reductase -- SDG secoisolariciresinol diglucoside -- ORF open reading frame -- GAPDH glyceraldehyde-3-phosphate dehydrogenase -- +P (+)-pinoresinol -- P (−)-pinoresinol -- ±L racemic lariciresinol -- (+)-/(−)-pinoresinol (+)- or (−)-pinoresinol -- S secoisolariciresinol -- HPLC high performance liquid chromatography; Botanical chemistry -- Periodicals; Biochemistry -- Periodicals; Botany -- Periodicals; Chimie végétale -- Périodiques; 572.2
• DOI: 10.1016/j.phytochem.2019.02.003
• Languages: English
• ISSNs: 0031-9422

Abstract

Abstract: Pinoresinol/lariciresinol reductase (PLR), an NADPH-dependent reductase that catalyzes the sequential reduction of pinoresinol into secoisolariciresinol via Lariciresinol, can lead to the structural and stereochemical diversity of lignans. The relationship between substrate-selective reaction of PLR and sequence homology still remains unclear. In this study, we focused on the contribution of the variable region between PLRs in determining substrate selectivity. Here, two Cs PLRs ( Cs PLR1 and Cs PLR2) were identified in the tea plant ( Camellia sinensis var. sinensis cv. Shuchazao). In vitro enzymatic assays showed that Cs PLR1 could convert (+)- and (−)-pinoresinol into lariciresinol or secoisolariciresinol, whereas Cs PLR2 catalyzed (+)-pinoresinol enantioselectively into (−)-secoisolariciresinol. Homology modeling and site-directed mutagenesis were used to examine the role of a variable loop in catalysis and substrate selectivity. The L174I mutant in Cs PLR1 lost the capacity to reduce either (+)- or (−)-pinoresinol but retained the ability to catalyze the reduction of (−)-lariciresinol. These findings provide a basis for better understanding of the substrate-selective reaction of PLR. Graphical abstract: Two Cs PLRs were identified in the tea plant ( Camellia sinensis ). Cs PLR1 converted (+)-/(−)-pinoresinol into lariciresinol or secoisolariciresinol, and Cs PLR2 catalyzed (+)-pinoresinol enantioselectively into (−)-secoisolariciresinol.Image 1 Highlights: Two Cs PLRs were identified in the tea plant ( Camellia sinensis ). CsPLR1 and CsPLR2 were highly expressed in fifth leaves and mature leaves. Cs PLR1 converted (+)-/(−)-pinoresinol into lariciresinol or secoisolariciresinol. Cs PLR2 catalyzed (+)-pinoresinol enantioselectively into (−)-secoisolariciresinol. A variable loop contributes to the reduction activity and substrate selectivity.