Arabidopsis 4-COUMAROYL-COA LIGASE 8 contributes to the biosynthesis of the benzenoid ring of coenzyme Q in peroxisomes

Abstract

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the β-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the β-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate β-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the β-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m−2 s−1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.

A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (coenzyme Q) in Arabidopsis

“…All together these results demonstrate that the loss of function of At1g24340 results in a metabolic blockage downstream of the formation of ubiquinone's ring, and confirm that At1g24340 acts as a ring modification enzyme. As previously observed for Arabidopsis mutants displaying similar losses in ubiquinone content (12,14), At1g24340 silenced plants were visually indistinguishable from their wild-type parent (data not shown).…”

The Mandelate Pathway, an Alternative to the Phenylalanine Ammonia Lyase Pathway for the Synthesis of Benzenoids in Ascomycete Yeasts

“…PAL catalyzes the conversion of phenylalanine (Phe) to trans-cinnamic acid, which is subsequently converted into benzaldehyde and other derived compounds through oxidative and nonoxidative pathways (1). In plants, PAL is involved in the biosynthesis of p-coumarate and kaempferol, both of which are precursors to 4-HB (17,18), and has been identified in some fungi of both Basidiomycota and Ascomycota, including Neurospora, Aspergillus, and Botrytis (14); however, this enzyme has not been identified in the Saccharomycotina subphylum. The formation of 4-HB in yeast and mammalian cells suggests that an unknown pathway exists (9).…”

Identification of the functions of 4-coumarate-CoA ligase/ acyl-CoA synthetase paralogs in potato

“…The substitution of the Cys from the box II motif towards a Trp would suggest these are not 4CL enzymes. However, recent reports on At4g19010 from subclade 3.4 (Block et al, 2014) and At5g38120 from subclade 3.3 (Soubeyrand et al, 2019) have unequivocally shown these to act as 4CL enzyme, albeit in a third pathway that, via the peroxisome leads to the synthesis of ubiquinone. Hence, we name these 4CL enzymes as class III 4CL enzymes.…”