A standard reaction condition and a single HPLC separation system are sufficient for estimation of monolignol biosynthetic pathway enzyme activities

Abstract

Lignin content and composition are largely determined by the composition and quantity of the monolignol precursors. Individual enzymes of the monolignol biosynthetic pathway determine the composition and quantity of monolignols. Monolignol biosynthesis in angiosperms is mediated by ten enzyme families. We developed a method using a total protein extract (soluble and microsomal) for the comprehensive and simultaneous analysis of these ten enzyme activities in a single target tissue, stem differentiating xylem (SDX) of Populus trichocarpa. As little as 300 mg fresh weight of SDX is sufficient for triplicate assays of all ten enzyme activities. To expand the effectiveness of the analysis, we quantified the reaction products directly by HPLC and developed a universal method that can separate the substrates and products of all enzymes. The specific activities measured with this simple approach are similar to those obtained with the optimum conditions previously established for each individual enzyme. This approach is applicable to the enzyme activity analysis for both P. trichocarpa (angiosperm) and Pinus taeda (gymnosperm) and is particularly useful when a large number of samples need to be analyzed for all monolignol biosynthetic enzymes.

Complete Proteomic-Based Enzyme Reaction and Inhibition Kinetics Reveal How Monolignol Biosynthetic Enzyme Families Affect Metabolic Flux and Lignin in Populus trichocarpa

“…Two homologs of CSE are present in the genome of P. trichocarpa (Vanholme et al, 2013). In contrast with all known steps in monolignol biosynthesis (Figure 1), where we have demonstrated strong enzymatic activity in SDX protein extracts (Liu et al, 2012), SDX protein extracts of P. trichocarpa do not show CSE activity (Figure 2A). No factor in P. trichocarpa SDX protein extracts could inhibit the CSE activity to cause a complete absence of this activity (Figures 2A and 2B) because such extracts have no effect on the Arabidopsis stem CSE activity (Figures 2B).…”

“…We first determined the Michaelis-Menten kinetic parameter (K m ), the turnover number (k cat ), and the catalytic efficiency (k cat /K m ) for all 21 enzymes (Figure 1) using optimized reaction conditions (Liu et al, 2012;Supplemental Table 3). The 24 monolignol biosynthetic pathway metabolites were obtained by chemical or biochemical synthesis or from commercial sources (Supplemental Methods 1).…”

Monolignol Pathway 4-Coumaric Acid:Coenzyme A Ligases in Populus. trichocarpa: Novel Specificity, Metabolic Regulation, and Simulation of Coenzyme A Ligation Fluxes

“…None of them were proteins known in the monolignol biosynthetic pathway (Shi et al, 2010;Liu et al, 2012). Given that monolignol proteins are highly abundant in SDX (Shi et al, 2010;Chen et al, 2011;Liu et al, 2012;Shuford et al, 2012aShuford et al, , 2012b, if they were phosphorylated, they most likely would have been detected. A similar phosphopeptide-enrichment/mass spectrometry (MS) strategy for the analysis of the phosphoproteome of dormant terminal buds in Populus simonii 3 Populus nigra identified 151 phosphoproteins .…”

“…Afterward, the reaction mixture was centrifuged for 20 min at 20,000g to condense the enzyme, and 40 mL of supernatant was injected onto a 4.6-3 150-mm Zorbax SB-C18 column (Agilent) for HPLC analysis. Conditions for HPLC analysis, including compound identification, were as described previously (Liu et al, 2012).…”

Comparative proteomic analysis of Populus trichocarpa early stem from primary to secondary growth

“…The extract concentration was determined using the Bradford method, and then it was used for enzyme assays. Caffeic acid 3-O-methyltransferase (COMT), caffeoyl coenzyme A O-methyltransferase (CCoAOMT) and cinnamyl alcohol dehydrogenase (CAD) activities in the extracts were determined as described in the literature [25,26]. The substrates and products of the enzyme reactions were separated on a Zorbax SB-C18 5 μm, 4.6 × 150-mm column (Agilent, Santa Clara, CA).…”