Biosynthesis of the Essential Respiratory Cofactor Ubiquinone from Phenylalanine in Plants

Abstract

The prenylated benzoquinone coenzyme Q or ubiquinone is a vital electron and proton carrier in the respiratory chain of mitochondria and some bacteria (Nowicka and Kruk, 2010Nowicka B. Kruk J. Occurence, biosynthesis and function of isoprenoid quinones.Biochim. Biophys. Acta. 2010; 1797: 1587-1605Crossref PubMed Scopus (316) Google Scholar). Due to its highly hydrophobic nature, ubiquinone can freely migrate in mitochondrial inner membrane carrying one or two electrons from either NADH dehydrogenase (complex I) or succinate dehydrogenase (complex II) to cytochrome bc1 complex (complex III). In plants, ubiquinone can additionally be reduced and oxidized by alternative NAD(P)H dehydrogenases and alternative oxidase, respectively. In this pathway, the reduction of ubiquinone is not obligatorily linked to the formation of proton gradient and thus is considered to give flexibility to mitochondrial electron transport chain under fluctuating conditions. Aside from its functions in the mitochondrial electron transport chain, ubiquinone is also considered to play a role as an anti/pro-oxidant and regulator of membrane fluidity in various cellular membranes. It is established that plants, like fungi and vertebrates, synthesize the prenyl moiety in mitochondria, where its conjugation to the benzenoid ring and the subsequent methylation and hydroxylation of the latter also occur (Figure 1A). However, the origin of the benzenoid backbone in plants was only revealed in a recent Arabidopsis study in which co-expression and knockout mutant analyses including feeding experiments with various substrates revealed that, within the peroxisome, the At4g19010 gene product activates the propyl chain of para-coumarate for its subsequent shortening by β-oxidation (Block et al., 2014Block A. Widhalm J.R. Fathi A. Cahoon R.E. Wamboldt Y. Elowsky C. Mackenzie S.A. Cahoon E.B. Chapple C. Dudareva N. et al.The origin and biosynthesis of the benzoid moiety of ubiquinone (Coenzyme Q) in Arabidopsis.Plant Cell. 2014; (in press).PubMed Google Scholar). Before the publication of this study, the plant route to the benzenoid backbone was unclear (Kawamukai, 2009Kawamukai M. Biosynthesis and bioproduction of coenyzme Q10 by yeasts and other organisms.Biotechnol. Appl. Biochem. 2009; 53: 217-226Crossref PubMed Scopus (78) Google Scholar) with knowledge from non-plant organisms hinting at a complex evolution of the pathway. In Escherichia coli, the ring of ubiquinone is made from 4-hydroxybenzoate via the direct aromatization of chorismate; however, this enzyme appears to be confined to eubacteria (Kawamukai, 2009Kawamukai M. Biosynthesis and bioproduction of coenyzme Q10 by yeasts and other organisms.Biotechnol. Appl. Biochem. 2009; 53: 217-226Crossref PubMed Scopus (78) Google Scholar). Saccharomyces cerevisiae is able to derive the ring either from 4-hydroxybenzoate or from the folate precursor para-aminobenzoate (pABA), which is prenylated and subsequently deaminated (Pierrel et al., 2010Pierrel F. Hamelin O. Douki T. Kieffer-Jaquinod S. Mühlenhoff U. Ozeir M. Lill R. Fontecave M. Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.Chem. Biol. 2010; 17: 449-459Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Vertebrates cannot synthesize pABA but rather appear to use 4-hydroxybenzoate in a linear pathway which links phenylalanine, tyrosine, and 4-hydroxybenzoate (Figure 1). That said, as is the case in yeast, the genes responsible for encoding the enzymes involved in the biosynthesis of 4-hydroxybenzoate remain to be identified. The situation in plants is further complicated by the fact that they do not convert phenylalanine into tyrosine most likely due to their lacking phenylalanine 4-hydroxylase (Pribat et al., 2010Pribat A. Noiriel A. Morse A.M. Davis J.M. Fouquet R. Loizeau K. Ravanel S. Frank W. Haas R. Reski R. et al.Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts.Plant Cell. 2010; 22: 3410-3422Crossref PubMed Scopus (35) Google Scholar). In order to address the question how is the benzenoid moiety of ubiquinone synthesized in plants?, Block et al., 2014Block A. Widhalm J.R. Fathi A. Cahoon R.E. Wamboldt Y. Elowsky C. Mackenzie S.A. Cahoon E.B. Chapple C. Dudareva N. et al.The origin and biosynthesis of the benzoid moiety of ubiquinone (Coenzyme Q) in Arabidopsis.Plant Cell. 2014; (in press).PubMed Google Scholar recently supplied U-13C-labeled phenylalanine, tyrosine, or pABA to axenic cultures of Arabidopsis. Both roots and shoots accumulated ring labeled 13C6 ubiquione following incubation in labeled phenylalanine or tyrosine, albeit accumulation of both was 10 times greater in the roots. By contrast, neither tissue accumulated ring labeled 13C6 ubiquinone in labeled pABA. Given the above stated inference that plants cannot convert phenylalanine into tyrosine, these data suggest that the formation of the benzenoid ring in plants might occur via two independent amino acid-fueled routes but that pABA is not incorporated into ubiquinone. The authors next searched the ATTED-II co-expression database (Obayashi et al., 2009Obayashi T. Hayashi S. Saeki M. Ohta H. Kinoshita K. ATTED-II provides coexpressed networks for Arabidopsis.Nucleic Acid Res. 2009; 37: D987-D991Crossref PubMed Scopus (306) Google Scholar), using key genes involved in the respiratory chain assembly as queries and found At4g19010 to be highly linked to these (see Figure 1). Furthermore, the gene exhibits a conserved domain found in 4-coumarate-CoA ligases and recombinant At4g19010 had previously been characterized as displaying CoA ligase activity. Further support for the function of the gene was provided by the fact that two of its strongest interactors in ATTED-II encode enzymes involved in the biosynthesis of ubiquinone. However, analysis of the corresponding T-DNA knockout line revealed that At4g19010 contributes to ring formation from phenylalanine but not tyrosine. To gain a more detailed insight into the exact pathway structure, wild-type and knockout plants were next fed with a range of compounds with results suggesting that t-cinnamate, p-coumarate, and 4-hydroxybenzoate are all part of the same branch-route to ubiquinone. Furthermore, this fact was supported by using the Arabidopsis ref3-2 mutant that displays reduced cinnamate 4-hydroxylase activity, and as such is compromised in its ability to convert t-cinnamate into p-coumarate. Consistently, the ref3-2 mutant was shown to contain only 40% of wild-type ubiquinone levels. Similarly, knockout lines of the PXA1 peroxisomal transporter also contained only around 45% of wild-type ubiquinone levels. When taken together, the data described above allowed the authors to postulate a convincing, if incomplete, metabolic route from phenylalanine to ubiquinone. Several other recent examples have employed similar approaches in order to identify and characterize unknown, yet crucial, steps in specialized metabolism. Indeed, similar approaches combining (either forward or reverse) genetics have recently identified several enzymes associated with aromatic amino acid biosynthesis and utilization (Dal Cin et al., 2011Dal Cin V. Tieman D.M. Tohge T. McQuinn R. de Vos R.C. Osorio S. Schmelz E.A. Taylor M.G. Smits-Kron M.T. Schurrink R.C. et al.Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit.Plant Cell. 2011; 23: 2738-2753Crossref PubMed Scopus (84) Google Scholar; Yoo et al., 2013Yoo H. Widhalm J.R. Qian Y. Maeda H. Cooper B.R. Jannasch A.S. Gonda I. Lewinsohn E. Rhodes D. Dudareva N. An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase.Nat. Commun. 2013; 25: 2833Google Scholar). In the first of these examples, overexpression of the petunia ODORANT transcription factor in tomato resulted in the identification of a suite of genes involved in the biosynthesis and further metabolism of phenylalanine (Dal Cin et al., 2011Dal Cin V. Tieman D.M. Tohge T. McQuinn R. de Vos R.C. Osorio S. Schmelz E.A. Taylor M.G. Smits-Kron M.T. Schurrink R.C. et al.Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit.Plant Cell. 2011; 23: 2738-2753Crossref PubMed Scopus (84) Google Scholar). These tomato genes include one encoding for prephenate aminotransferase that converts prephenate into arogenate, thus completing the arogenate pathway of phenylalanine biosynthesis. As in the above example, feeding of 13C labeled phenylalanine was used to further confirm the pathway structure. In the second example, it was demonstrated that plants utilize a microbial-like phenylpyruvate pathway to produce phenylalanine, and that flux through this route increases in compensation when that through the arogenate pathway is restricted. It was also shown that the plant phenylpyruvate pathway utilizes a cytosolic aminotransferase that links the coordinated catabolism of tyrosine to serve as the amino donor, thus interconnecting the extra-plastidial metabolism of these amino acids. This discovery as such uncovers yet another level of complexity in the plant aromatic amino acid regulatory network. Looking more broadly, co-expression analyses combined with analysis of knockout mutants have also been used to identify critical transporters including a monolignol and a glycerate:glycolate transporter. The lignol transporter was identified by co-expression analysis of the ABCG transporter (Alejandro et al., 2012Alejandro S. Lee Y. Tohge T. Sudre O. Osorio S. Park J. Bovet L. Lee Y. Gelner N. Fernie A.R. et al.AtABCG29 is a monolignol transporter involved in lignin biosynthesis.Curr. Biol. 2012; 10: 1207-1212Abstract Full Text Full Text PDF Scopus (216) Google Scholar). Given that members of this sub-family have been shown to transport a broad range of fatty acids and terpenoids, the authors asked the question whether this class of transporter could also be implicated in the transport of phenolic compounds. The results revealed that AtABCG29/PDR1 exhibited a high co-expression ratio with three genes of the phenylpropanoid biosynthesis pathway, which is involved in the synthesis of lignin and flavonoids. The well-correlated genes correspond to two 4-coumarate coenzymeA (CoA) ligases (4CL2 and 4CL5) and one caffeoyl CoA-O-methyltransferase. Moreover, seven further genes related to phenylpropanoid biosynthesis are co-expressed with AtABCG29, albeit with lower co-expression ratios. Biochemical analyses of knockout mutants similar to that carried out in the Block paper, although independent of labeling studies, confirmed that this gene encoded a monolignol transporter. The similarly elusive glycerate:glycolate transporter was identified in a similar pathway-based manner—albeit this time also utilizing labeling studies (Pick et al., 2013Pick T.R. Bräutigam A. Schulz M.A. Obata T. Fernie A.R. Weber A.P. PLGG1, a plastidic glycolate glycerate transporter, is required for photorespiration and defines a unique class of metabolite transporters.Proc. Natl Acad. Sci. U S A. 2013; 110: 3185-3190Crossref PubMed Scopus (119) Google Scholar). These techniques are thus clearly effective methods for the identification of the genes encoding missing metabolic or transport steps. The (partial) characterization of the phenylpropanoid derived benzenoid biosynthetic pathway represents an elegant example of the power of this approach. Hopefully, the tyrosine derived pathway and the steps linking p-coumaryl-CoA and 4-hydroxybenzoate will also be resolved shortly, providing the final pieces in the jigsaw of the plant pathway of ubiquinone biosynthesis.