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MiniReview

Ubiquinone biosynthesis in microorganisms

R. Meganathan *

Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA Received 22 May 2001; received in revised form 3 July 2001; accepted 3 July 2001

First published online 2 August 2001

Abstract

The quinoid nucleus of the benzoquinone, ubiquinone (coenzyme Q; Q), is derived from the shikimate pathway in bacteria and

eukaryotic microorganisms. Ubiquinone is not considered a vitamin since mammals synthesize it from the essential amino acid tyrosine.

Escherichia coliand other Gram-negative bacteria derive the 4-hydroxybenzoate required for the biosynthesis of Q directly from chorismate.

The yeast,Saccharomyces cerevisiae, can either form 4-hydroxybenzoate from chorismate or tyrosine. However, unlike mammals,S.

cerevisiaesynthesizes tyrosine in vivo by the shikimate pathway. While the reactions of the pathway leading from 4-hydroxybenzoate to Q

are the same in both organisms the order in which they occur differs. The 4-hydroxybenzoate undergoes a prenylation, a decarboxylation

and three hydroxylations alternating with three methylation reactions, resulting in the formation of Q. The methyl groups for the

methylation reactions are derived fromS-adenosylmethionine. While the prenyl side chain is formed by the 2-C-methyl-

D-erythritol

4-phosphate (non-mevalonate) pathway inE. coli, it is formed by the mevalonate pathway in the yeast. ß 2001 Federation of European

Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords:Ubiquinone; Coenzyme Q; Q biosynthesis; Isoprenoid; Mevalonate; Non-mevalonate

1. Introduction

The isoprenoid quinones of the benzene and naphtha- lene series are widespread in microorganisms. Aerobic Gram-negative bacteria and eukaryotes contain the benzo- quinone, ubiquinone, as the sole quinone, while the facul- tative anaerobic bacteria such asEscherichia colicontain the naphthoquinones, demethylmenaquinone (DMK) and menaquinone (MK) in addition to ubiquinone [1]. The archaea, as a group, lack ubiquinone. In bacteria and eu- karyotes, including the fungi, ubiquinone functions as an electron carrier in the aerobic respiratory chain. This MiniReview is concerned with the biosynthesis of the benzoquinone, ubiquinone (coenzyme Q). According to the IUPAC commission on nomenclature, both names are recognized. In this MiniReview and in biosynthetic studies, the newer ubiquinone is generally used, while in biomedical studies, coenzyme Q is used. Abbreviations are QorQ-nwhere `n' refers to the number of prenyl units present in the side chain (Fig. 1).E. colicontains Q-8

(n=8, a 40 carbon isoprenoid side chain) as the predom-inant quinone with minor amounts of Q-1 to Q-7 and Q-9

[1]. The predominant side chain length is a constant de- pending on the species. Thus, for example,Gluconobacter suboxydans,Rhodobacter capsulatus,E. coli, and the yeast, Saccharomyces cerevisiaehave side chain lengths ofn=10,

9, 8, and 6, respectively.

2. Structure and precursors

The structure of ubiquinone (4) is shown in Fig. 1. The quinonoid nucleus is derived from the shikimate pathway via chorismate (1) in bacteria or tyrosine (2) in higher eukaryotes [2^5]. The eukaryotic microorganism,S. cere- visiae, can synthesize Q from either chorismate (1) or ty- rosine (2) [4,5]. While in yeast and probably other fungi the tyrosine is derived from chorismate in vivo, it has to be provided as an essential amino acid for mammals, which lack the shikimate pathway and, hence, Q is not consid- ered a vitamin. The methyl groups on the benzene ring are derived fromS-adenosylmethionine (SAM). In eukaryotes, the isopentenyl diphosphate required for the formation of the polyprenyl side chain is derived from acetate via the mevalonate pathway. In bacteria, it is formed by the newly discovered 2-C-methyl-

D-erythritol 4-phosphate (MEP)

0378-1097/01/$20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

PII: S0378-1097(01)00330-5

* Tel.: +1-(815) 753 7803; Fax: +1-(815) 753 0461. E-mail address:rmeganathan@niu.edu (R. Meganathan).

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pathway also known as the 1-deoxy-D-xylulose 5-phos- phate pathway, or the non-mevalonate pathway [20,21]. Much of the early work on the pathway for the biosyn- thesis of the quinonoid nucleus was the result of studies onE. coliby Gibson, Young and colleagues [6,7] using isotopic tracer methodology. It was shown that [G- 14 C]- shikimate was incorporated into Q, establishing that the quinone is a product of the shikimate pathway. In subse- quent studies,E. coliwas subjected to chemical mutagen- esis and screened for mutants unable to grow on oxidiz- able substrates, such as succinate and malate, while retaining the ability for fermentative growth on glucose. The Q biosynthetic intermediates accumulated by these mutants were extracted in organic solvents and puri¢ed by thin-layer chromatography. The structures of these in- termediates were determined by mass spectrometry and nuclear magnetic resonance spectrometry. The role and location of the intermediates in the pathway were estab- lished by enzymatic analysis of the mutants coupled with genetic analysis by transduction. In this review, the work onE. coli, a representative of bacteria, and the yeastS. cerevisiae, a representative of eukaryotic microbes, will be discussed. Due to the space constraints of the journal, only reviews, recent papers, and a few selected references will be cited. For a complete list of references, the reader is referred to the literature cited in the review articles. How-

ever, it should be pointed out that these reviews are spe-cialized and address speci¢c aspects of Q biosynthesis. Forexample, there are separate reviews covering the biosyn-

thesis of the quinoid ring from chorismate inE. coli, the reaction mechanisms for the various enzymes, the biosyn- thesis of isopentenyl diphosphate by the mevalonate path- way in eukaryotes and the MEP pathway in bacteria and the path of assembly of the polyprenyl side chain. This MiniReview brings together all these pathways succinctly so as to provide a guide to further in depth reading. The biosynthesis of the `quinonoid' head of Q is ¢rst discussed. This is followed by a description of the two pathways for the biosynthesis of dimethylallyl diphosphate (DMAPP) and/or isoprenyl diphosphate (IPP), and ¢nally, the conversion of IPP into the side chain (`tail') precursor, polyprenyl diphosphate (PPP).

3. Biosynthesis of the quinonoid ring

The biosynthesis of the quinonoid ring and the various ring modi¢cation reactions inE. colihave been reviewed [8,9]. A mechanistic perspective on the various reactions has been provided [10,11].

3.1. Biosynthesis of 4-hydroxybenzoate in E. coli

The formation of 4-hydroxybenzoate (3) from choris- mate (1) is the ¢rst committed step in the biosynthesis of Q. This aromatizing reaction is catalyzed by the enzyme chorismate pyruvate-lyase encoded by theubiCgene. Mu- tants blocked in this conversion have been isolated and characterized. TheubiCmutants are de¢cient in the for- mation of Q and are characterized by their inability to grow aerobically on oxidizable substrates such as succi- nate. TheubiCgene has been overexpressed, and the en- zyme puri¢ed to homogeneity and characterized.

3.2. Biosynthesis of 4-hydroxybenzoate in yeast

Yeast seems to produce 4-hydroxybenzoate by two dif- ferent ways. It may be produced directly from chorismate by the chorismate pyruvate-lyase reaction similar toE. coli, or alternately from tyrosine, similar to higher eukary- otes. Consistent with the existence of two di¡erent routes is the fact that yeast mutants blocked in the formation of

4-hydroxybenzoate have never been isolated. Evidence for

the presence of the two alternate routes was obtained us- ing shikimate pathway mutants. Mutants blocked in the formation of shikimate or chorismate are expected to be de¢cient in the formation of Q, due to their inability to form 4-hydroxybenzoate. Addition of tyrosine to the growth medium restored the ability of these mutants to form 4-hydroxybenzoate and Q. It was further shown that wild-type yeast normally uses the conversion of cho- rismate (1) to 4-hydroxybenzoate (3) as the source of pre- cursor for Q. However, in the shikimate pathway mutants, Fig. 1. Biosynthetic precursors of ubiquinone.1, chorismate;2, tyro- sine;3, 4-hydroxybenzoate;4, ubiquinone; SAM,S-adenosylmethio- nine; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphos- phate; MEP, 2-C-methyl-

D-erythritol 4-phosphate.

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tyrosine (2) is able to compensate fully by providing the

4-hydroxybenzoate (3) required for Q biosynthesis [4,5]. It

seems likely that many of the lower eukaryotes that have the shikimate pathway may contain dual pathways for the biosynthesis of 4-hydroxybenzoate (3). In animal cells,

4-hydroxybenzoate (3) is formed from the essential amino

acid tyrosine (2). A pathway for the conversion of tyrosine (2) to 4-hydroxybenzoate (3) was proposed by Booth et al. [12] following urinary excretion studies on animals admin- istered with various phenolic acids. Based on these studies and on the incorporation of radiolabeled phenolic acids into Q by liver and yeast, the following pathway was pro- posed (Fig. 2). TyrosineC4-hydroxyphenylpyruvateC4- hydroxybenzoate. The in vitro conversion of tyrosine (2) to 4-hydroxyphenylpyruvate (5) and its subsequent reduc- tion to 4-hydroxyphenyllactate (

6) has been shown in ex-

tracts of rat liver and yeast. Further reactions involved in the conversion of 4-hydroxyphenyllactate (

6) to 4-hydroxy-

benzoate (3) are not known. However, it has been sug- gested that 4-hydroxyphenyllactate (6) is converted to

4-hydroxycinnamate (7), followed byL-oxidation as the

coenzyme A derivative, resulting in the formation of 4- hydroxybenzoate (3) [4]. Evidence in support of this path- way has recently been obtained in higher plants [13] (Fig. 2).

4. Reactions of ring modi¢cation inE. coli

4.1. Prenylation

The prenylation of 4-hydroxybenzoate (3) to 3-octa- prenyl-4-hydroxybenzoate (

11) is carried out by the mem-

brane bound enzyme 4-hydroxybenzoate octaprenyltrans-

ferase inE. coli(Fig. 3). The enzyme is non-speci¢c andcan use a variety of prenyl diphosphates as side chainprecursors. The lack of speci¢city of the enzyme also ex-

tends to the aromatic substrate. Thus, the enzyme tolerates substitutions by various groups at di¡erent positions on the benzene ring of Q. The length of the prenyl side chain is a constant for each organism. This constancy of length is determined by the availability of the prenyl diphosphate in the cell (see Sections 7 and 8).

4.2. Decarboxylation

The 3-octaprenyl-4-hydroxybenzoate (11) is decarboxy- lated to 2-octaprenylphenol (12) by the enzyme 3-octa- prenyl-4-hydroxybenzoate decarboxylase. InSalmonella entericaandE. coli, two genes designated asubiDand ubiXencode the two decarboxylases carrying out this re- action. The open reading frames ( orf) encoding these two enzymes have been recently identi¢ed [11,14^16].

4.3. Hydroxylations and methylations

The 2-octaprenylphenol (

12) undergoes three hydroxy-

lations alternating with three methylation reactions result- ing in the formation of ubiquinol (20) and then Q (4). For convenience, the hydroxylations are considered together followed by the methylation reactions.

4.3.1. Hydroxylations

Three monooxygenases are involved in the introduction of three hydroxyl groups at positions C-6, C-4, and C-5 of the benzene ring, respectively.

The three reactions are:

1. 2-octaprenylphenol (12)C2-octaprenyl-6-hydroxyphe-

nol (13);

2. 2-octaprenyl-6-methoxyphenol (16)C2-octaprenyl-6-

methoxy-1,4-benzoquinol ( 17);

3. 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol

benzoquinol (19). Mutants de¢cient in each of the hydroxylations have been isolated and designated asubiB(12C13),ubiH (16C17), andubiF(18C19), respectively. The open read- ing frames encoding these reactions have been identi¢ed [11,17,18]. These mutants accumulate the intermediate be- fore the block, namely (

12), (16), (18) respectively.

When wild-typeE. coliis grown anaerobically on the oxidizable substrate glycerol with fumarate as an electron acceptor, it forms considerable quantities of Q (50^70% of aerobically grown cells). Mutants blocked in the non-hy- droxylating reactions of the pathway, such asubiA (3C11),ubiD(11C12), andubiE(17C18), are Q de¢- cient under aerobic as well as anaerobic conditions, thus establishing that the same genes and enzymes are involved Fig. 2. Proposed pathway for the biosynthesis of 4-hydroxybenzoate from tyrosine.2, tyrosine;5, 4-hydroxyphenylpyruvate;6, 4-hydroxy- phenyllactate;7, 4-hydroxycinnamate orp-coumarate;8,p-coumaroyl-

CoA;9, 4-hydroxybenzoyl-CoA;3, 4-hydroxybenzoate.

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in both conditions. However, the mutants blocked in the oxygenases discussed above, namelyubiB,ubiH, andubiF, synthesize Q under anaerobic conditions, providing evi- dence that there are alternate hydroxylases for the syn- thesis of Q anaerobically. These anaerobic hydroxylases likely derive the hydroxyl groups from the solvent water [8,11].

4.3.2. Methylations

There are three methylation reactions in the pathway, ofwhich two are onOand one onC. These reactions are as

follows:

1. 2-octaprenyl-6-hydroxyphenol (

13)C2-octaprenyl-6-

methoxyphenol (

16) (i.e.O-methylation);

2. 2-octaprenyl-6-methoxy-1,4-benzoquinol (

17)C2-oc-

taprenyl-3-methyl-6 methoxy-1,4-benzoquinol (

18) (i.e.

C-methylation);

3. 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzo-

quinol (19)Cubiquinol (20) (i.e.O-methylation).

Fig. 3. Ubiquinone biosynthetic pathway inE. coliand yeast. It should be noted that the chemical numbering system locates the prenyl side chain in

compound11at the C-3 carbon and in compounds14and15at the C-5 carbon. In compounds12,13,16and subsequent intermediates, the prenyl

side chain is assigned to C-2. Compounds17^20are drawn in the quinol form. Some authors draw these structures in the quinone form. The chemical

names for the intermediates of the pathway are as follows:1, chorismate;2, tyrosine;3, 4-hydroxybenzoate;11, 3-polyprenyl-4-hydroxybenzoate;12,

2-polyprenylphenol;13, 2-polyprenyl-6-hydroxyphenol;14, 3,4-dihydroxy-5-polyprenylbenzoate;15, 3-methoxy-4-hydroxy-5-polyprenylbenzoate;16,2-

polyprenyl-6-methoxyphenol;17, 2-polyprenyl-6-methoxy-1,4-benzoquinol;18, 2-polyprenyl-3-methyl-6-methoxy-1,4-benzoquinol;19, 2-polyprenyl-3-

methyl-5-hydroxy-6-methoxy-1,4-benzoquinol;20, ubiquinol;Q, ubiquinone. The conversion of20, ubiquinol, toQis thought to be non-enzymatic.

ForE. coli, polyprenyl=octaprenyl; for yeast, polyprenyl=hexaprenyl. The genes forE. coliare theubigenes and the yeast genes are theCOQgenes.

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As mentioned above, these three methylations alternate with hydroxylations, introducing methyl groups at the 6- OH, at the ring C-3, and at the 5-OH respectively.S-Ad- enosylmethionine is the actual methyl donor being con- verted toS-adenosylhomocysteine (SAH). A non-speci¢cC-methyltransferase encoded by theubiE gene methylates the ring C-3 resulting in the conversion of (17C18). In addition this methyltransferase also methyl- ates demethylmenaquinone to menaquinone.

BothO-methylations mentioned above, namely the

methylation of 6-OH and 5-OH, are carried out by the sameO-methyltransferase encoded by theubiGgene. This non-speci¢cO-methyltransferase, in addition to car- rying out the methylation of (13C16) and (19C20) also carries out theO-methylation of theS. cerevisiaeQ bio- synthetic intermediate (

14C15) [11,16].

5. Reactions of ring modi¢cation in yeast

In yeast, the side chain precursor used for the prenyl- transferase is hexaprenyl diphosphate. The product of the reaction 3-hexaprenyl-4-hydroxybenzoate (

11) undergoes

further ring modi¢cation reactions in a di¡erent order than that ofE. colijust described. Thus, compound (11) ¢rst undergoes hydroxylation to (14) followed by methyl- ation to (15) and ¢nally decarboxylation to (16) (Fig. 3). TheO-methylase encoded by the COQ3 gene like UbiG is non-speci¢c and it catalyzes the conversion of (

14C15)

and (19C20). In addition, it converts theE. coliQ bio- synthetic intermediate (13C16). TheE. coliUbiG can function in vivo provided the protein contains the yeast mitochondrial import leader sequence [11,16].

6. Biosynthesis of the polyprenyl tail precursors

The biosynthetic precursors for the polyprenyl tail are isopentenyl diphosphate (IPP) (26) and DMAPP (27). There are two di¡erent pathways for the biosynthesis of these precursors (Fig. 4). Those bacteria that possess Q appear to use the MEP pathway (Fig. 4B), while the eu- karyotic microorganisms use the mevalonate pathway (Fig. 4A) [19,20]. However, there are exceptions; certain eukaryotic microbes, like the green algae and the malarial parasitePlasmodium falciparum, appear to possess the MEP pathway, while a few bacteria contain the mevalo- nate pathway [21]. These exceptions to the general distri- bution have been attributed to horizontal gene transfer. Complete lists of the distribution of the pathways in var- ious microorganisms are available [22,23].

6.1. MEP pathway

Two glycolytic intermediates, pyruvate (28) and

D-glyc-eraldehyde 3-phosphate (30), are the starting precursors for the biosynthesis of IPP (

26) and DMAPP (27) by the

MEP pathway (Fig. 4B). The pyruvate is decarboxylated in a TPP-requiring reaction by a mechanism analogous to the pyruvate dehydrogenase (E 1 ) of the pyruvate dehydro- genase complex. The product of decarboxylation, hy- droxyethyl-TPP anion (29), condenses with the aldehyde group of

D-glyceraldehyde 3-phosphate (30), resulting in

the formation of 1-deoxy-

D-xylulose 5-phosphate (DXP)

(31). The enzyme catalyzing the reaction is DXP synthase encoded by thedxsgene located at 9 min on theE. coli linkage map. In the next step of the pathway, the DXP undergoes a benzylic type rearrangement and reduction resulting in the formation of MEP (32). This conversion is mediated by an NADPH-dependent reductoisomerase encoded by thedxr gene located at 4.2 min on theE. colilinkage map.

Further studies established that the MEP (

32) is con-

verted to 4-diphosphocytidyl-2-C-methylerythritol (33)in a novel CTP-dependent reaction. The enzyme catalyzing the reaction, designated as 4-diphosphocytidyl-2-C-meth- ylerythritol synthase, is encoded by theispDgene. Subse- quently, compound (33) is phosphorylated at the 2-posi- tion by an ATP-dependent kinase encoded by theispE gene resulting in the formation of compound (

34). In the

next step of the pathway, an enzyme designated as 2-C- methyl-

D-erythritol 2,4-cyclodiphosphate synthase en-

coded by theispFgene eliminates CMP from compound (34) resulting in the formation of 2-C-methyl-

D-erythritol

2,4-cyclodiphosphate (35) [11,21]. It is worth mentioning

that compound (35) had been shown to accumulate in bacteria under oxidative stress [11]. The genes, enzymes, intermediates and reactions in- volved in the conversion of the cyclodiphosphate (35)to IPP (26) and DMAPP (27) are yet to be determined. How- ever, evidence has been obtained in support of the biosyn- thesis of both IPP (26) and DMAPP (27) by independent mechanisms in the terminal steps of the pathway [21,24]. An IPP isomerase encoded byidigene interconverts IPP (26) and DMAPP (27). It has been postulated that these terminal steps for the formation of IPP (

26) and DMAPP

(27) from the cyclic diphosphate (35) may involve a ring opening reaction, two dehydrations and two reduction steps [21]. It is likely that the recently identi¢edlytB gene ofE. coliis involved in one of the unidenti¢ed late reactions just mentioned [25].

6.2. Mevalonate pathway

Eukaryotic microorganisms like the fungi and yeasts lack the MEP pathway and rely on the mevalonate path- way with the few exceptions mentioned above (Fig. 4A). The starting precursor for the mevalonate pathway is ace- tyl-CoA. The pathway is initiated by the transfer of an acetyl group from one acetyl-CoA (

10) to the methyl car-

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