Báo cáo khoa học: Biosynthesis of D-arabinose in mycobacteria – a novel bacterial pathway with implications for antimycobacterial therapy pdf

Wolucka Laboratory of Mycobacterial Biochemistry, Institute of Public Health, Brussels, Belgium Keywords cell wall biosynthesis; D -ribose; ethambutol; Mycobacterium tuberculosis; mycoli

Biosynthesis of D -arabinose in mycobacteria – a novel

bacterial pathway with implications for antimycobacterial therapy

Beata A Wolucka

Laboratory of Mycobacterial Biochemistry, Institute of Public Health, Brussels, Belgium

Keywords

cell wall biosynthesis; D -ribose; ethambutol;

Mycobacterium tuberculosis; mycolic acid;

polyisoprenoid glycolipid; review

Correspondence

B A Wolucka, Laboratory of Mycobacterial

Biochemistry, Institute of Public Health, 642

Engeland Street, B-1180 Brussels, Belgium

(b-d-arabinofuranosyl-1-O-monophospho-to decaprenyl-phospho-arabinose, which is a substrate for rases in the synthesis of the cell-wall arabinogalactan and lipoarabinomannanpolysaccharides of mycobacteria The first step of the proposed decaprenyl-phospho-arabinose biosynthesis pathway in Mycobacterium tuberculosis andrelated actinobacteria is the formation of d-ribose 5-phosphate from sedohep-tulose 7-phosphate, catalysed by the Rv1449 transketolase, and⁄ or the isom-erization of d-ribulose 5-phosphate, catalysed by the Rv2465 d-ribose5-phosphate isomerase d-Ribose 5-phosphate is a substrate for the Rv1017phosphoribosyl pyrophosphate synthetase which forms 5-phosphoribosyl1-pyrophosphate (PRPP) The activated 5-phosphoribofuranosyl residue ofPRPP is transferred by the Rv3806 5-phosphoribosyltransferase to decaprenylphosphate, thus forming 5¢-phosphoribosyl-monophospho-decaprenol Thedephosphorylation of 5¢-phosphoribosyl-monophospho-decaprenol to deca-prenyl-phospho-ribose by the putative Rv3807 phospholipid phosphatase isthe committed step of the pathway A subsequent 2¢-epimerization of decapre-nyl-phospho-ribose by the heteromeric Rv3790⁄ Rv3791 2¢-epimerase leads tothe formation of the decaprenyl-phospho-arabinose precursor for the synthe-sis of the cell-wall arabinans in Actinomycetales The mycobacterial 2¢-epimer-ase Rv3790 subunit is similar to the fungal d-arabinono-1,4-lactone oxidase,the last enzyme in the biosynthesis of d-erythroascorbic acid, thus pointing to

arabinosyltransfe-an evolutionary link between the d-arabinofurarabinosyltransfe-anose- arabinosyltransfe-and l-ascorbic related pathways Decaprenyl-phospho-arabinose has been a lead compoundfor the chemical synthesis of substrates for mycobacterial arabinosyltransfe-rases and of new inhibitors and potential antituberculosis drugs The peculiar(x,mono-E,octa-Z) configuration of decaprenol has yielded insights into lipidbiosynthesis, and has led to the identification of the novel Z-polyprenyldiphosphate synthases of mycobacteria Mass spectrometric methods weredeveloped for the analysis of anomeric linkages and of dolichol phosphate-related lipids In the field of immunology, the renaissance in mycobacterialpolyisoprenoid research has led to the identification of mimetic mannosyl-b-1-phosphomycoketides of pathogenic mycobacteria as potent lipid antigenspresented by CD1c proteins to human T cells

acid-Abbreviations

ALO, D -arabinono-1,4-lactone oxidase; Araf, D -arabinofuranose; GLO, L -gulono-1,4-lactone oxidase; PRPP, 5-phosphoribosyl 1-pyrophosphate.

The family of mycobacteria comprises about 100

species, several of which are pathogens of humans

and⁄ or animals, including Mycobacterium tuberculosis,

M bovis, M leprae, M avium-intracellulare, M ulcerans

and M marinum The pathogenic mycobacteria are

inherently resistant to many antibacterial drugs and

can persist for years inside infected cells

Mycobacte-rium tuberculosis, the aetiological agent of tuberculosis,

kills about 1.7 million people per year [1] and,

accord-ing to World Health Organization estimations, is

pres-ent in a latpres-ent form in about one-third of the world’s

population (http://www.who.int/tb/en) A combination

of several factors, such as the requirement of long-term

multidrug therapy for the treatment of tuberculosis,

the synergy between M tuberculosis and human

immu-nodeficiency virus infections [2], the emergence of

mul-tidrug-resistant strains and, in particular, the recent

outbreaks of extensively drug-resistant tuberculosis

[3,4], has contributed to the persistence of tuberculosis

as a global public health problem

Several existing antituberculosis drugs, including the

first-line drugs isoniazid and ethambutol, act at the

level of the cell wall This vital structure plays a crucial

role in the virulence and pathogenicity of M

tuberculo-sis Mycobacteria possess a thick, highly impermeable

hydrophobic cell wall composed of a thin layer of

pep-tidoglycan, d-arabinofuranose (Araf)-containing

arabi-nogalactan and arabinomannan polysaccharides,

mannans, glucans, long-chain (C70–C90) a-branched,

b-hydroxy fatty acids (mycolic acids) and other lipids,

glycolipids, poly-l-glutamate–glutamine polymers,

enzymes and other proteins Like teichoic acids in

other Gram-positive bacteria [5], arabinogalactan is

covalently attached to peptidoglycan by a

phosphodi-ester linkage The arabinan part of arabinogalactan is,

in turn, esterified to mycolic acids, thus forming a

pep-tidoglycan–arabinogalactan–mycolic acid skeleton

(reviewed in [6]) This rigid model of the mycobacterial

cell wall is now being replaced by a more dynamic

pic-ture, in which the cell wall undergoes substantial

modi-fications in response to changing growth conditions, as

may occur in host cells, for example, after the

proposed transfer from phagosomal to cytosolic

com-partments [7] The plasma membrane-anchored

lipo-arabinomannans and lipomannans, reminiscent of

lipoteichoic acid, are probably translocated to the

outer layer of the cell wall and processed to lipid-free

arabinomannans and mannans [8] The presence, at

least transient, of different proteins and enzymes in the

M tuberculosis cell wall, such as the porins that are

involved in the transport of hydrophilic molecules

[9,10], the catalase-peroxidase katG [11,12], the heat

shock protein 60 chaperones (GroEL1) that assist lipid

traffic [13,14], the antigen 85 mycolyltransferases [15]complexed with a histone-like protein [16], the gluta-mine synthetase involved in the synthesis of poly-l-glutamate–glutamine polymers [17], serine⁄ threonineprotein kinases [18–20] and other virulence factors[21,22], points to the dynamic structure, and suggests

an active role of the organelle in host–pathogen actions Indeed, profound alterations of the cell-wallcomposition are thought to occur that could lead toantigenic variation [23] and isoniazid resistance [24] ofnon-replicating, dormant M tuberculosis found in per-sistent infections Moreover, during human infection,the pathogen elaborates new macromolecular struc-tures at the cell surface: pili, putative host colonizationfactors [25]

inter-d-Arabinose occurs rarely in nature In contrast with

d-arabinopyranose, which is found in some eukaryotes,such as trypanosomatids and plants, Araf is confined

to the prokaryotic world, where it is a constituent ofcell-surface polymers and glycolipids In mycobacteriaand related Actinomycetales species, Araf is a compo-nent of the arabinan parts of the arabinogalactan and(lipo)arabinomannan polymers of the cell wall and ofsome glycerol-based glycolipids [26] The branchedarabinan chains of the arabinogalactan are attached tothe linear galactan backbone The arabinan consists of

an inner linear region of Araf-(1fi 5)-a-Araf and ofbranched non-reducing terminal Ara6 motifs: Arafb1

fi 2Arafa1 fi 5(Arafb1 fi 2Arafa1 fi 3)Arafa1 fi5Arafa1 About two-thirds of the terminal b-Arafand the penultimate 2-a-Araf serve as attachmentsites for mycolic acids (reviewed in [6])

The arabinan part of the M tuberculosis binomannan consists of linear segments of Araf-(1fi 5)-a-Araf with some a(1 fi 3) branching Thenon- reducing termini are composed of two distinctmotifs: the Ara6 motif similar to that present in arabi-nogalactan, and a simplified linear Ara4 motif: Ara-

lipoara-fbfi 2Arafa1 fi 5Arafa1 fi 5Arafa1 Some of thenon-reducing arabinofuranose termini are capped withshort chains of a(1fi 2) d-mannose [27]

The physiological role of arabinans was thought to

be exclusively structural and of similar importancewithin Corynebacterineae (the mycobacteria⁄ nocar-dia⁄ corynebacteria group); however, recent studies havechallenged this simplistic view For example, arabinan-devoid mutants of corynebacteria can be obtained[28,29], whereas abrogation of arabinan synthesis islethal in mycobacteria In addition, the complex regula-tion [30] and functions [31] of arabinan-assemblingEmb proteins suggest that this polymer could play arole in sensing mechanisms and possibly other pro-cesses, in particular in pathogenic mycobacteria

Despite the efforts of many research groups, the

bio-synthesis of d-arabinose in mycobacteria was an

enigma for many years until the isolation of decaprenyl

-phospho-arabinose and its decaprenyl-phospho-ribose

precursor in 1990, and the proposal of the last step of

d-arabinose synthesis catalysed by a 2¢-epimerase

(Scheme 1) [32] The subsequent structural

charac-terization of both the

b-d-arabinofuranosyl-1-monophosphodecaprenol (Fig 1B) [33] and the

b-d-ribofuranosyl-1-monophosphodecaprenol (Fig 1C)

[34] allowed the biological origins of bacterial Araf to

be deciphered, and a new era in the study of cell-wallbiosynthesis in mycobacteria to be started

The discovery: arabinose, decaprenyl-phospho-ribose and other endogenous lipid-linked sugars of mycobacteria

decaprenyl-phospho-In spite of several claims of the existence of activatednucleotide and 1-phosphate derivatives of d-arabinose[35–37], water-soluble activated forms of d-arabinose,

Scheme 1 The original scheme of biosynthesis of D -arabinofuranosyl residues of the cell-wall arabinogalactan and lipoarabinomannan in mycobacteria, including a feedback mechanism and possible sites of action of ethambutol, an antituberculosis drug [32] Two possible sites

of ethambutol are indicated: 1, inhibition of arabinosyltransferase activity; 2, inhibition of certain step(s) in the biosynthesis of the acceptor X, where X may be a polyprenyl-pyrophosphoryl-oligosaccharide or a growing polymer chain Note that option 2, namely the inhibition of arab- inan synthase activity (Emb), was demonstrated later by others (see text and Fig 2) Araf, D -arabinofuranose; Ribf, D -ribofuranose.

Fig 1 Decaprenyl phosphate and

decapre-nyl-phospho-monosaccharides of

myco-bacteria (A) The mycobacterial lipid carrier

C 50 -decaprenyl phosphate has a unique

stereoconfiguration and contains only one

trans (E)-isoprene residue at its x-end [33]

(see Fig 3) (B)

Decaprenyl-phospho-arabi-nose, the only known D -arabinose donor for

the synthesis of the cell-wall arabinogalactan

and lipoarabinomannan in mycobacteria

[32,33] (C) Decaprenyl-phospho-ribose, the

direct precursor of the b- D

-arabinofuranosyl-monophosphodecaprenol donor (B) and the

major form of the naturally occurring

deca-prenyl-phospho-sugars of mycobacteria

[32,34] (D) The mycobacterial

decaprenyl-phospho-mannose, a minor component

[107].

such as d-arabinose phosphates and d-arabinose

nucle-otides, have never been demonstrated in mycobacteria

Exogenously added d-arabinose is catabolized by a

spontaneous M smegmatis mutant via an inducible,

fungal-like pathway [32,38,39] that converts an

aldo-pentose into a ketoaldo-pentose [40] (Fig 2) In the

myco-bacterial pathway, d-arabinose is reduced by a

NADPH-dependent d-arabinose dehydrogenase to

d-arabinitol, and the latter compound is oxidized to

d-xylulose by a NAD-dependent d-arabinitol genase d-Xylulose can then be phosphorylated to

dehydro-d-xylulose 5-phosphate and enter the pentose phate cycle [32,39] In contrast with mycobacteria, themajority of bacteria use either isomerase⁄ kinase oroxidation pathways for the utilization of pentoses[41,42] Interestingly, the oxidation of d-arabinose to

phos-d-arabinono-1,4-lactone does not occur in

mycobacte-ria [32,39], but in fungi, where it has been believed, at

least until recently [43], to be involved in the

biosyn-thesis of d-erythroascorbic acid [44]

After a fruitless search for water-soluble

intermedi-ates of d-arabinose, we looked for lipid-linked

pyro-phospho-oligosaccharides similar to the dolichol-linked

oligosaccharides of archaebacteria [45] Indeed,

gradi-ent-eluted DEAE-cellulose fractions of organic extracts

from M smegmatis contained lipid-linked

galactose-oligosaccharides, but also large amounts of

mono-charged, acid-labile arabinose, ribose and mannose

linked to phosphorylated isoprenoid lipids, although

some mycolic acids could be detected as well

Subse-quent analysis of the monocharged glycolipids by

fast-atom bombardment mass spectrometry demonstrated

the presence of decaprenyl-phospho-pentoses and

deca-prenyl phosphate ions at m⁄ z 909 and m ⁄ z 777,

respec-tively [32] This was the beginning of a fruitful search

that has led to the identification of the d-arabinose

pathway, and to a better understanding of cell-wall

bio-synthesis and of the mechanism of action of ethambutol

in mycobacteria In particular, we discovered that

eth-ambutol does not interfere with

decaprenyl-phospho-arabinose synthesis, and that the site of action of the

drug is downstream in the arabinan pathway [32]

Accordingly, it was proposed that: (a)

decaprenyl-phos-pho-arabinose is synthesized via a 2¢-epimerization of

decaprenyl-phospho-ribose, and serves as the donor of

d-arabinofuranosyl residues in the biosynthesis of the

cell-wall arabinogalactan and (lipo)arabinomann; (b)

ethambutol inhibits an arabinosyltransferase or an

arabinan-forming enzyme, and this inhibition results in

the accumulation of decaprenyl-phospho-arabinose in

mycobacteria; (c) the synthesis of the

decaprenyl-phos-pho-ribose precursor is controlled by a feedback

mecha-nism (Scheme 1) [32] These conclusions have proven to

be correct and have served as the basis for further

research

The details of the decaprenyl-phospho-arabinosestructure, including the determination of the absoluteconfiguration, anomeric linkage and ring form of the

d-arabinosyl residue, were solved later using combinedproton-NMR spectroscopy, gas chromatography andmass spectrometry (Fig 1B) [33] NMR analysis alsoallowed the determination of the particular structure

of the mycobacterial decaprenol with important cations regarding its biosynthesis (Figs 1A and 3) Itwas a big surprise for us to find that what is lacking inthe 10 isoprene unit-containing C50-decaprenol ofmycobacteria is not a cis (Z)-unit, but one of the twotrans (E)-isoprene units that are localized at the x-end

impli-of the known polyisoprenyl lipid carriers, including thecommon bacterial undecaprenol The proposedx,mono-E,octa-Z configuration of the mycobacterialdecaprenol [33] was, in fact, the first hint of the exis-tence of unusual Z-prenyl diphosphate synthases inmycobacteria: a Z-farnesyl diphosphate synthase thatwould provide an x,E,Z-farnesyl diphosphate for asubsequent specific enzyme, a Z-decaprenyl diphos-phate synthase These unusual enzymes have beenidentified recently (see below)

The structure of the endogenous syl-1-monophosphodecaprenol of mycobacteria wassolved (Fig 1B) This was unprecedented because,until that time, no other natural lipid-linked sugar iso-lated from an organism had been fully structurallycharacterized [46,47]

b-d-arabinofurano-The next step was the structural elucidation of prenyl-phospho-ribose (Fig 1C) [34] The presence ofsubstantial amounts of decaprenyl-phospho-ribose waspuzzling because no ribose-containing polymers haveever been described in mycobacteria We proposed thatdecaprenyl-phospho-ribose is converted to decaprenyl-phospho-d-arabinose by a novel 2¢-epimerase ofmycobacteria (Scheme 1) [32] The decaprenyl-phospho-ribose 2¢-epimerase has been identifiedrecently

deca-Fig 2 The metabolism of D -arabinose in mycobacteria The fungal-like assimilation pathway for D -arabinose of Mycobacterium smegmatis [32,39] is shown (top reactions) Decaprenyl-phospho- D -arabinose, the only known D -arabinofuranose donor, and decaprenyl-phospho-ribose (in rectangles), were isolated from M smegmatis [32] and structurally characterized (see Fig 1) Decaprenyl-phospho-arabinose was pro- posed to be synthesized via a 2¢-epimerization of decaprenyl-phospho-ribose, and to control the synthesis of the latter compound by a feed- back mechanism The heteromeric decaprenyl-phospho-ribose 2¢-epimerase (Rv3790 ⁄ Rv3791) was identified recently Ethambutol, a first-line drug for the treatment of tuberculosis, inhibits the utilization of decaprenyl-phospho-arabinose [32,33] at the level of the Emb proteins that are involved in the formation of arabinans [75,88] The enzymatic steps leading from the well-known 5-phosphoribosyl 1-pyrophosphate (PRPP) intermediate to the formation of decaprenyl-phospho-ribose were identified later by in vitro assays D -Ribose 5-phosphate, the direct precursor of PRPP, is proposed to be synthesized mainly by an essential transketolase (Rv1449) of the non-oxidative pentose phosphate pathway A possible involvement of a non-essential ribose 5-phosphate isomerase (Rv2465) and of the oxidative pentose phosphate pathway enzymes is also shown Intermediates of the fungal-like catabolic pathway are shown in green; the non-oxidative and oxidative parts of the pentose phosphate pathway are shown in blue and violet, respectively; the decaprenyl-phospho-arabinose pathway is shown in red Essen- tial genes of M tuberculosis, as determined by Himar1-based transposon mutagenesis [52,133], are indicated in bold, and cloned genes are underlined.

The discovery of decaprenyl-phospho-ribose pointed

to the involvement of activated ribose derivatives in

the biosynthesis pathway to d-arabinose This

observa-tion was crucial for the identificaobserva-tion of the precursor

of decaprenyl-phospho-ribose An obvious candidate

to test as a donor of the activated d-ribofuranosyl

resi-due was the well-known, high-energy bond-containing

intermediate for nucleotide synthesis: 5-phosphoribosyl1-pyrophosphate (PRPP) In vitro assays of crudemembranes of M smegmatis incubated with [14C]-labelled PRPP and synthetic decaprenyl phosphate assubstrates demonstrated the synthesis of decaprenyl-phospho-ribose 5¢-phosphate, which, on dephosphory-lation, produces decaprenyl-phospho-ribose [37] The

Fig 3 The biosynthesis of C 50 -decaprenyl pyrophosphate in Mycobacterium tuberculo- sis The particular structure of the mycobac- terial decaprenol (see Fig 1A) implies the existence in mycobacteria of unique Z-prenyl diphosphate synthases that use x,E-geranyl pyrophosphate as a substrate The non- essential Rv1086 Z-farnesyl diphosphate synthase and the essential Rv2361 Z-deca- prenyl diphosphate synthase have been identified [71,72].

progress of the mycobacterial genome sequencing

pro-jects [48,49] has allowed a comparative genomics

approach that has led to the identification of the

mycobacterial decaprenyl-phospho-ribose 2¢-epimerase

and the phosphoribosyl transferase, involved in the

biosynthesis of decaprenyl-phospho-arabinose [50] and

Synthesis ofD-ribose 5-phosphate

The first step in the biosynthesis of the

b-d-arabino-furanosyl-1-O-monophosphodecaprenol

(decaprenyl-phospho-arabinose) in mycobacteria (Fig 2) is the

synthesis of d-ribose 5-phosphate d-Ribose

5-phos-phate could be synthesized by an amphibolic, thiamine

(vitamin B1) diphosphate-dependent transketolase

(sedoheptulose 7-phosphate:d-glyceraldehyde

3-phos-phate glycolaldehydetransferase; EC 2.2.1.1), which

reversibly transfers a keto group from sedoheptulose

7-phosphate to d-glyceraldehyde 3-phosphate, and

produces d-ribose 5-phosphate and d-xylulose

5-phos-phate, according to reaction (1):

sedoheptulose 7-phosphateþD-glyceraldehyde 3-phosphate

,D-ribose 5-phosphateþD-xylulose5-phosphate ð1Þ

The transketolase is a ubiquitous enzyme that links the

glycolytic and pentose phosphate pathways, but has

never been studied in mycobacteria The M

tuberculo-sisgenome contains one sequence encoding a putative

transketolase (Rv1449), and the gene is essential [52]

Otherwise, d-ribose 5-phosphate could be formed

from another intermediate of the pentose phosphate

pathway, d-ribulose 5-phosphate, by the ribose

5-phos-phate isomerase (Rv2465) (Fig 2) Surprisingly, the

ribose 5-phosphate isomerase, and also several other

pentose phosphate pathway genes, such as d-xylulose

5-phosphate 3-epimerase (Rv1408) and the

6-phos-phoglucono-1,5-lactone lactonase (Rv1445), are

app-arently not essential in M tuberculosis [52]

Consequently, the reaction catalysed by the ribose

5-phosphate isomerase probably plays a minor role in

the synthesis of the vital arabinans in mycobacteria

Formation of 5-phosphoribosyl-a-1-pyrophosphate

The second step in the biosynthesis of

decaprenyl-phospho-arabinose (Fig 2) is the reaction of ribose

5-phosphate with ATP to yield pyrophosphate and AMP, catalysed by a PRPPsynthetase (ribose 5-phosphate diphosphokinase;

5-phosphoribosyl-a-1-EC 2.7.6.1) (reaction 2):

ribose 5-phosphateþ ATP , 5-phospho-a-D

-ribose 1-pyrophosphateþ AMP ð2ÞPRPP is a key metabolite in the purine and pyrimidinenucleotide de novo and salvage pathways, the biosynthe-sis of pyridine nucleotide coenzymes and the synthesis

of histidine and tryptophan By analogy with thedecaprenyl-phospho-arabinose biosynthesis of myco-bacteria, PRPP is proposed to be a precursor of b-d-ribofuranosyl residues of lipopolysaccharides andcapsular polysaccharides of Gram-negative bacteria,such as Pseudomonas aeruginosa, Salmonella sp., Shigellasp., Escherichia coli, Proteus sp., Haemophilus influenzaeand, perhaps, of eukaryotic trypanosomatids [34].Mycobacterium tuberculosis contains one PRPP syn-thetase protein (Rv1017) that shares at least 43% iden-tity with its human, plant and bacterial homologues.The mycobacterial PRPP synthetase sequence contains

a conserved PRK03092 domain from Val227 toAla240 (VLIDDMIDTGGTIA) that corresponds tothe PRPP binding motif The PRPP synthetases areknown to undergo a complex regulation, and bothADP and inorganic phosphate (Pi) are the known allo-steric regulators of the enzyme [53] In spite of its cen-tral role in cell-wall, nucleic acid and proteinbiosynthesis, the mycobacterial PRPP synthetase hasnot yet been characterized

In Fig 2, we propose that the inhibition of arabinansynthesis by ethambutol, and the resulting accumula-tion of decaprenyl-phospho-arabinose [32,33], couldhave further repercussions via a feedback mechanism,and inhibit, directly or indirectly, the PRPP synthetaseactivity in mycobacteria This would result indecreased amounts of the PRPP precursor and, inagreement with the observed complex effects of thedrug, lead to the inhibition of the synthesis of decapre-nyl-phospho-ribose [32,33], but also of nucleic acidsand other compounds [54]

Synthesis of b-Dphodecaprenol

-5¢-phosphoribosyl-1-monophos-The next step of the decaprenyl-phospho-arabinosepathway (Fig 2) is the reversible transfer of the5-phosphoribosyl residue from the activated PRPPdonor to the decaprenyl phosphate acceptor, catalysed

by a 5-phospho-a-d-ribose 1-pyrophosphate:decaprenylphosphate 5-phosphoribosyltransferase (reaction 3):

5-phospho-a-D-ribose 1-pyrophosphate

þdecaprenyl phosphate , 50-phosphoribosyl

b-d-5¢-phosphoribosyl-1-monophosphodeca-prenol Thus, the reaction would occur with an

inversion of the anomeric configuration of the

5-phos-phoribosyl residue, although direct evidence is lacking

The decaprenyl phosphate-dependent

phosphoribosyl-transferase activity was demonstrated in vitro using

crude membranes from M smegmatis and a [14

C]-labelled PRPP substrate [37] It was claimed that

poly-prenylphosphate-5-phosphoarabinose was one of the

reaction products and the direct precursor of

polypre-nyl-phospho-arabinose in mycobacteria, and it was

concluded that the epimerization at the C2 position of

the ribosyl residue takes place at the level of either

phosphoribose pyrophosphate or

polyprenylphosphate-5-phosphoribose [36,37]

The M tuberculosis genes encoding

5-phospho-a-d-ribose 1-pyrophosphate:decaprenyl phosphate

5-phos-phoribosyltransferase (Rv3806) and the downstream

enzyme decaprenyl-phospho-ribose 2¢-epimerase (Rv3790 ⁄

Rv3791) were identified only recently using a

compara-tive genomics strategy, as suggested earlier [34], namely

by searching M tuberculosis orthologues of the

Azorhizobium genes that are involved in the

d-arabi-nosylation of nodulation factor glycolipids [50,51] It is

worth noting that the sequences of the Nod-factor

genes for d-arabinosylation have never been published,

and the gene functions are, in fact, unknown [51] In

addition, the early work reported the presence of

d-arabinose in the Azorhizobium Nod factor glycolipids

in the pyranose rather than furanose form [55], and

convincing evidence for the presence of Araf is lacking

In contrast, the advent of the M tuberculosis and

M lepraegenome data [48,49] has played an

indisput-able role in the identification of genes for the

myco-bacterial arabinogalactan⁄ arabinomannan synthesis,

and led to the proposed d-arabinose pathway in

myco-bacteria

Homologues of the Rv3806 protein (annotated as

UbiA prenyltransferases) are present in some Archaea

and in many eubacteria, such as mycobacteria,

coryne-bacteria and nocardia that share a similar composition

of their cell walls, certain species of cyanobacteria,

gamma-proteobacteria, clostridia and others The

Rv3806 phosphoribosyltransferase (302 amino acids) is

an integral membrane protein that requires Mg2+ for

its activity The unpurified recombinant enzyme

pres-ent in the membrane of the E coli host had apparpres-ent

Kmvalues for PRPP and the decaprenyl phosphate ofplant origin substrates of 120 and 22 lm, respectively[51] The enzyme had a preference for medium-chainpolyprenyl phosphates (C50–C55) and showed no activ-ity with a short-chain C20-polyprenyl phosphate The

pH optimum for the phosphoribosyltransferase tion was pH 7.5–8 Contrary to the authors’ claim[51], the reaction catalysed by the 5-phospho-a-d-ribose 1-pyrophosphate:decaprenyl phosphate 5-phos-phoribosyltransferase is probably not the committedstep of decaprenyl-phospho-arabinose biosynthesis,because it is reversible in the absence of pyrophospha-tase activity

reac-Synthesis of b-Dnol (decaprenyl-phospho-ribose)

-ribosyl-1-monophosphodecapre-Decaprenyl-phospho-ribose is the major form of thelipid-linked pentoses in mycobacteria [34] (Fig 1C) It

is formed by the removal of a 5¢-phosphate group ofthe b-d-5¢-phosphoribosyl-1-monophosphodecaprenolprecursor, catalysed by a phosphatase (reaction 4):

50-phosphoribosyl-b-1-monophospho-decaprenol

! b-D-ribosyl-1-monophospho-decaprenolþ Pi ð4ÞThe phosphatase reaction is expected to be irreversible,and thus it would represent the committed step in thebiosynthesis of decaprenyl-phospho-arabinose in myco-bacteria Inspection of the M tuberculosis operonsinvolved in the biosynthesis of the arabinan and galac-tan polymers has revealed the presence of an unknownPAP2-family phospholipid phosphatase (Rv3807),which is located next to the phosphoribosyltransferase(Rv3806) discussed above The Rv3807 orthologues arepresent in all Corynebacterineae The Rv3807 protein

is therefore a good candidate for a specific phospho-ribose-5¢-phosphate phosphatase Surpris-ingly, the Rv3807 gene is apparently not essential [52],whereas all the other genes related to decaprenyl-phos-pho-arabinose synthesis are annotated as essentialgenes Further studies are necessary to elucidate thebiological function of the Rv3807 gene product

decaprenyl-2¢-Epimerization of decaprenyl-phospho-ribose todecaprenyl-phospho-arabinose

The last step of the biosynthetic pathway of nyl-phospho-arabinose (b-d-arabinofuranosyl-1-mono-phosphodecaprenol) is the 2¢-epimerization of

decapre-d-ribofuranosyl to d-arabinofuranosyl at the level ofdecaprenyl-phospho-pentoses, as originally proposed

[32,33] (Fig 2) This conversion proceeds via a

decaprenyl-phospho-2¢-keto-d-arabinose intermediate,

which is probably not released from the mycobacterial

enzyme under physiological conditions (reaction 5):

b-D-ribofuranosyl-1-monophosphodecaprenol

! ½20-keto-b-D

-arabinofuranosyl-1-monophosphodecaprenol ! b-D

The decaprenyl-phospho-ribose 2¢-epimerase is a

het-eromeric enzyme composed of two types of

polypep-tide that are annotated as an oxidoreductase and a

short-chain dehydrogenase⁄ reductase, and encoded by

the Rv3790 and Rv3791 genes, respectively, of the

M tuberculosis genome [50] The exact composition of

the enzyme is unknown However, simultaneous

expression of both polypeptides is required for

epimer-ase activity Close homologues of the Rv3790 and

Rv3791 proteins are present in arabinan-synthesizing

mycobacteria, corynebacteria, nocardia and related

actinobacteria, but also in other bacteria, many of

which are pathogens and symbionts of animals and

plants: for example, Pseudomonas aeruginosa,

Burk-holderiasp., Legionella pneumophila, Leptospira

interro-gans and Rhizobium etli Interestingly, species that are

known to contain Araf as a component of their

lipo-polysaccharide, such as the opportunistic pathogen

Pseudomonas aeruginosa and the legume symbiont

Sinorhizobium meliloti, possess sequences that are

simi-lar (35% identity) to the Rv3790 and Rv3791 subunits

of the heteromeric 2¢-epimerase of M tuberculosis

The Rv3790 oxidoreductase protein (461 amino

acids) contains a FAD-binding N-terminal domain and

a C-terminal d-arabinono-1,4-lactone oxidase (ALO)

signature from T423 to L458 The ALO domain is

characteristic for l-gulono-1,4-lactone oxidase

(GLO)-like enzymes that catalyse the last step in the

biosyn-thesis of l-ascorbic acid (or its 5-carbon homologue

d-erythroascorbic acid) in plants, animals, fungi and

some microbes [56,57] The Rv3790 protein shares

22% identical residues with d-arabinono-1,4-lactone

oxidase of Sacccharomyces cerevisiae (ALO1) [58] The

protein also shows a limited identity at both the

N- and C-termini (26% and 38% identity, respectively)

with the recently identified l-gulono-1,4-lactone

dehy-drogenase (Rv1771) of M tuberculosis [59] The yeast

ALO1 enzyme catalyses the last step of oxidation of

d-arabinono-1,4-lactone to d-erythroascorbic acid, and

uses molecular oxygen as electron acceptor The

Rv1771 dehydrogenase is probably involved in the

syn-thesis of l-ascorbic acid (vitamin C) in M tuberculosis;

the enzyme is specific for l-gulono-1,4-lactone, and

can use both cytochrome c and a phenazine derivative

as electron acceptors [59]

The d-arabinono-1,4-lactone substrate of the yeastALO1 enzyme has a furan-based ring structure that issimilar to the d-arabinofuranosyl moiety of the epim-erase reaction product (Fig 5) Although the mecha-nism of GLO and other GLO-like enzymes is not wellunderstood, the GLO-catalysed reaction is thought toproceed via oxidation of the 2-hydroxyl group to a2-keto derivative, which subsequently undergoes anenolization to form l-ascorbic acid (or d-erythroascor-bic acid) It is probable therefore that the Rv3790 sub-unit(s) is directly responsible for the conversion ofdecaprenyl-phospho-ribose to the corresponding2¢-keto-b-d-erythropentofuranose derivative (Figs 2and 5)

In conclusion, little is known about the Rv3790⁄Rv3791 decaprenyl-phospho-ribose 2¢-epimerase of

M tuberculosis In particular, the nature of the flavincofactor of the Rv3790 subunit and of the electron ac-ceptors has not been elucidated

As discussed above, an evolutionary link existsbetween Araf and l-ascorbic acid⁄ d-erythroascorbicacid biosynthesis pathways An ancestor GLO-likegene of Actinomycetales or an unrelated gene that hasacquired an ALO-like domain by convergent evolutioncould evolve into an Araf synthesizing enzyme(Rv3790) by recruiting an ancient short-chain dehydro-genase⁄ reductase (Rv3791) that reduces the 2¢-keto

d-arabinofuranose ring to a d-arabinofuranosyl due Interestingly, pathogenic actinobacteria, including

resi-M tuberculosis, resi-M bovis, resi-M ulcerans and resi-M num, have acquired, via gene duplication⁄ divergentevolution or horizontal gene transfer, another GLOgene (Rv1771 in M tuberculosis) for the synthesis of

mari-l-ascorbic acid (or a related compound) The product

of the Rv1771-catalysed reaction might interfere with

l-ascorbic acid-dependent signal transduction ways of animal hosts; however, its functions in

path-M tuberculosisare still unknown [59]

Decaprenyl-phospho-arabinose 1-monophosphodecaprenol) is the only known donor

(b-d-arabinofuranosyl-of d-arabinofuranosyl units in the synthesis ofarabinans of Actinomycetales Disruption of the geneencoding the 5-phospho-a-d-ribose 1-pyrophos-phate:decaprenyl phosphate 5-phosphoribosyltransfer-ase (UbiA) produces a d-arabinose-deficient mutant ofCorynebacterium glutamicum that is devoid of the cell-wall arabinan–corynomycolic acid complex [28] Thissurprising result indicates that both the arabinan part

and the bound corynomycolic acids of the cell-wall

peptidoglycan–arabinogalactan–corynomycolate core

are not essential for the survival of C glutamicum In

contrast, the arabinan part of the

peptidoglycan–arabi-nogalactan–mycolate core is essential in mycobacteria,

because disruption of the priming arabinosyltransferase

AftA (Rv3792), which adds the first

d-arabinofurano-syl residue to the galactan core, or of the Rv3806

phosphoribosyl transferase, is lethal in M tuberculosis

[28,60]

Mycobacterium smegmatis synthesizes an additional

compound containing an activated d-arabinose

resi-due, namely a partially saturated

b-d-arabinosyl-1-monophospho-octahydroheptaprenol (Fig 4B) [61]

The biosynthesis of the C35-isoprenyl-phospho

deriva-tive of d-arabinose is unknown, although it is possible

that the compound is synthesized via the

decaprenyl-phospho-arabinose pathway because of the low

speci-ficity of the decaprenyl-phosphate-dependent enzymes

Otherwise, the C35arabinose could be synthesized by a direct transfer ofthe d-arabinofuranosyl unit from decaprenyl-phospho-arabinose or another donor to the C35-octahydrohep-taprenyl phosphate acceptor In agreement with thelatter proposal, ribosylated derivatives of C35-octahy-droheptaprenyl phosphate have never been described.The biological function of b-d-arabinosyl-1-mono-phospho-octahydroheptaprenol of M smegmatis isunknown Moreover, it is not clear whether othermycobacteria synthesize C35-octahydroheptaprenyl-phosphate derivatives

-octahydroheptaprenyl-phospho-Interestingly, single terminal d-arabinofuranosylresidues of short lipoarabinomannans of C glutami-cum are apparently not derived from decaprenyl-phos-pho-arabinose, but rather from another, still unknown,donor [62]

In contrast with Araf, which is present exclusively inbacteria, d-arabinopyranose is found in polysaccharides

Fig 4 The partially and fully saturated cosylated phospholipids of mycobacteria (A) The major form of the lipid-linked mannose

gly-in Mycobacterium smegmatis, the partially saturated short-chain C35-octahydrohepta- prenyl-phospho-mannose [107] (B) A minor form of the lipid-linked D -arabinose of

M smegmatis, the partially saturated chain C35-octahydroheptaprenyl-phospho- arabinose [61] (C) The mycolylated isopren- oid phospholipid of M smegmatis [120] (D) The C30-mannosyl-b-1-phosphomycoketide

short-of M avium (E) A similar C 34 derivative of

M tuberculosis [121] The compounds in (D) and (E) are not related to polyisoprenoids, and are synthesized in pathogenic mycobac- teria by a polyketide synthase [108].