ATP, the cell's energy currency, is largely produced in mitochondria, membrane-bound bodies within cells that couple ATP formation to the controlled burn of carbohydrates or lipids (fat). Coenzyme Q (also known as ubiquinone or CoQ) is a complex organic molecule essential for mitochondrial energy production. CoQ is also a crucial antioxidant that can protect membrane lipids from oxidative damage. The CoQ molecule has two parts: (1) a long "tail" that anchors it in the membrane, and (2) an aromatic ring structure (benzoquinone) with properties that allow it to ferry electrons and protons essential to ATP formation. Cells are generally thought to synthesize the benzoquinone ring from the precursor 4-hydroxybenzoic acid (4-HB). Although nine genes essential for CoQ biosynthesis have been identified in yeast, plants and animals, none are required for 4-HB synthesis. Therefore, major questions remain unanswered regarding the generation of the aromatic benzoquinone component of CoQ. 4-HB has been believed to be synthesized in bacteria and yeast via coumarate from the phenylpropanoid biosynthesis pathway, a pathway used by plants to produce a wide variety of metabolites, or in animals by using the amino acid tyrosine. Recent observations indicate that yeast can also use 4-aminobenzoic acid (pABA) as a ring precursor in CoQ biosynthesis. This is surprising because pABA is a well-known precursor of folate, one of the B vitamins, but has not previously been connected with CoQ. The utilization of pABA as a ring precursor represents a novel biosynthetic pathway for the production of CoQ. This research utilizes yeast to define how pABA is converted to CoQ, how tyrosine is converted to 4-HB, and how coumarate is converted to 4-HB. This research, therefore, addresses the basic biochemistry required for energy generating processes in living cells. Results may also lead to applications in agriculture and medicine, as the enzymes involved are potential targets for both herbicides and antimicrobials.
Broader Impacts: This project affords undergraduate students, particularly underrepresented minority students, the opportunity to do cutting edge research. UCLA undergraduate students generated the results identifying pABA as a ring precursor to CoQ biosynthesis in yeast. Construction and characterization of yeast deletion mutants, characterization of yeast growth, determination of CoQ content, isotopic labeling of yeast cultured in different types of minimal media, and preparation and HPLC analyses of yeast lipid extracts are all carried out by undergraduate students working on independent research projects. The PI is Chair of the Diversity and Leadership Committee in the UCLA Department of Chemistry and Biochemistry, and both the PI and Co-PI are active in advising and mentoring women and under-represented minority undergraduate and graduate students.
Scientific Merit Coenzyme Q (ubiquinone, CoQ or Q) (Figure 1), is a lipid component of our cells. Q has two major functions in cells, it is essential for producing energy in the form of ATP, the cell’s energy currency and Q is also an antioxidant that functions to protect other membrane lipids from peroxidation. Q consists of two parts: (1) A long "tail" that derives from the isoprenoid biosynthetic pathway, where n designates the number of isoprene units. For example, yeast produce CoQ6 with a hexaprenyl tail, humans produce CoQ10 with a decaprenyl tail. (2) The benzoquinone "ring" of CoQ is responsible for transport of protons and electrons. The reversible transition of Q from the oxidized quinone (Q) to the reduced hydroquinone (QH2) (Figure 2) enables Q to function in energy production and as an antioxidant (Figure 3). Our studies focused on determining how cells synthesize the "ring" of Q. Our work established that the benzoquinone "ring" of Q derives from distinct aromatic ring precursors, including para-aminobenzoic acid (pABA) (Figure 4). This was a very surprising finding because pABA was thought to be a committed precursor in the synthesis of folates (vitamin B9). Our discovery was also surprising because pABA contains a nitrogen substituent on the ring, whereas Q contains no nitrogen. We showed that the biosynthesis of Q from pABA requires the same set of Coq polypeptides that convert 4-hydroxybenzoic acid (4HB) to Q (Figure 4). We showed that the Coq9 polypeptide is required to remove the nitrogen substituent from the ring. We also identified ring precursors that "bypass" certain steps in Q biosynthesis. Use of these precursors helps identify the functional roles of some of the enzymes (Coq polypeptides) needed to synthesize Q. Our work demonstrated that biosynthesis of Q is accomplished by a multi-subunit complex termed the CoQ Synthome (Figure 5). The CoQ synthome is composed of multiple Coq polypeptides and is stabilized by the presence of Q itself. The formation or maintenance of the CoQ synthome depends on the Coq8 polypeptide, and its presence is required to observe modified forms of several of the Coq polypeptides (certain Coq polypeptides are decorated with phosphate groups). We showed that over-expression of the Coq8 polypeptide, a putative kinase, restores the steady state levels of several of the other Coq polypeptides, and helps to diagnose certain blocked steps in yeast coq mutants. Our discovery of new pathways in yeast coenzyme Q biosynthesis sets the stage for elucidating whether similar pathways operate in other types of cells, including human, animal, plant and microbes. Acquiring this knowledge is required to understand how coenzyme Q content can be regulated under different physiological conditions. Broader Impacts Undergraduate and Graduate Student Training and Professional Development This project fosters activities that include: the training of graduate students, their development as teachers and mentors of undergraduate students, and supporting undergraduate students as active participants in the scientific discovery process. Students start out tackling very basic, fundamental problems of metabolism, and quickly become "hooked" as they formulate questions that involve yeast and E. coli genetics, molecular and cell biology, and lipid biochemistry. Initially undergraduate students work closely with a graduate student mentor. After undergraduates gain experience with sterile techniques and culturing bacteria and yeast, they perform assays of cell growth and phenotype analyses, and then proceed with more sophisticated projects. Undergraduate researchers gain hands-on understanding of how metabolic pathways are elucidated. They learn that what is reported in the textbook may not be the only pathway, and that there may be other routes yet to be discovered. They are excited to participate in the discovery process, and help assemble figures and prepare posters for presentations at conferences and manuscripts for publication. Thirty-one undergraduate students worked on this project, and eighteen of these students are either co-authors on published papers, or manuscripts in preparation. Impact on disciplines in other fields Our work is expanding the classical role of pABA in folate and one carbon metabolism to include essential energy metabolism and other roles of Q. These results may lead to applications in agriculture and medicine, because the enzymes utilizing pABA are potential targets for herbicides and antimicrobials. In addition to their effects on folate metabolism, pABA mimics may act to inhibit Q biosynthesis, and this may explain some of the side effects of antifolates. Understanding the role of alternate ring precursors in coenzyme Q biosynthesis may lead to the development of strategies that could be used to augment the content of Q in cells. These results may lead to applications in agriculture and medicine, because the enzymes utilizing some of the alternate ring precursors are potential targets for herbicides and antimicrobials.
