Mechanism of action of nucleoside antibacterial natural product antibiotics

ABSTRACT This article reviews the structures and biological activities of several classes of uridine-containing nucleoside antibiotics (tunicamycins, mureidomycins/pacidamycins/sansanmycins,

liposidomycins/caprazamycins, muraymycins, capuramycins) that target translocase MraY on the peptidoglycan biosynthetic pathway. In particular, recent advances in structure-function

studies, and recent X-ray crystal structures of translocase MraY complexed with muraymycin D2 and tunicamycin are described. The inhibition of other phospho-nucleotide transferase enzymes

related to MraY by nucleoside antibiotics and analogues is also reviewed. You have full access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS

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TEREPHTHALIC ACID ANTIBIOTICS Article Open access 05 November 2024 The discovery of the liposidomycin nucleoside antibiotics by Isono et al. in 1985 [1], and the nucleoside antibiotic

tunicamycin by Takatsuki et al. [2] has led to the identification and study of a related collection of uridine-containing nucleoside antibiotics with potent antibacterial activity,

targetting the enzyme phospho-MurNAc-pentapeptide translocase (MraY) on the peptidoglycan cell wall biosynthetic pathway. The structures of each family have been reviewed in detail in

reviews in 2003 [3] and 2010 [4]. This review will discuss recent structure-activity studies on each group of nucleoside antibiotics, and the mechanism of inhibition of translocase MraY, in

particular, the recent crystal structures of nucleoside antibiotics bound to MraY. ANTIBACTERIAL NUCLEOSIDE ANTIBIOTICS TARGETTING BACTERIAL PEPTIDOGLYCAN BIOSYNTHESIS THE TUNICAMYCIN GROUP

OF GLCNAC-TUNICAMINE NUCLEOSIDE ANTIBIOTICS (TUNICAMYCINS, STREPTOVIRUDINS, CORYNETOXINS) The tunicamycin group of nucleoside antibiotics were isolated in 1971 from _Streptomyces

lysosuperficus_ by Takatsuki et al. [2]. They contain a uracil base attached to a C11 tunicamine sugar, glycosylated at C11 by a GlcNAc sugar and N-acylated at C10 by a C12–C15 fatty acid

(see Fig. 1). They showed antibacterial activity against a range of Gram-positive bacteria, especially those in the _Bacillus_ genus (MIC 0.1–20 µg/ml) [2], but also showed toxicity towards

eukaryotic cells, due to inhibition of eukaryotic N-linked glycoprotein biosynthesis [5]. The streptovirudins and corynetoxins contain the same uracil-tunicamine skeleton, but are acylated

by different fatty acids [3]. The biosynthetic gene cluster for the tunicamycin antibiotics has been identified in _Streptomyces chartreusis_ [6], and the biosynthetic pathway has been shown

to involve an unusual radical SAM enzyme TunM in the assembly of the tunicamine sugar [7]. A total synthesis of tunicamycin V was reported in 2017 by Yamamoto et al. [8], which has enabled

the synthesis of tunicamycin analogues for structure-activity study [9]. A lipid-truncated analogue and an analogue lacking the GlcNAc sugar both lost 1000-fold in MraY inhibition activity

but retained some enzyme inhibition, while an analogue lacking the nucleoside base was completely inactive [9]. The presence of the uracil base has been shown to be required in other

nucleoside antibiotic families [3, 4], which can be rationalised by the MraY structural studies described in section ‘Structure of _Aquifex aeolicus_ MraY and its complexes with nucleoside

antibiotics'. THE MUREIDOMYCIN GROUP OF UREIDYL PEPTIDE NUCLEOSIDE ANTIBIOTICS (MUREIDOMYCINS, PACIDAMYCINS, NAPSAMYCINS, SANSANMYCINS) Mureidomycins A-D were isolated from

_Streptomyces flavidoviridens_ SANK 60486, and first reported in 1989 [10]. They showed potent antimicrobial activity against a range of _Pseudomonas_ strains (MIC 0.1–3 µg/ml), and

protected mice against infection by _Pseudomonas aeruginosa_ (ED50 50 mg/kg for MrdC) [11, 12]. Phospho-MurNAc-pentapeptide translocase (MraY) on the bacterial peptidoglycan biosynthetic

pathway was identified as the molecular target of these compounds [11]. A closely related series of pacidamycins 1–7, isolated from _Streptomyces coeruleorubidus_ strain AB 1183F-64, were

also reported in 1989 [13,14,15]. The pacidamycins also showed antimicrobial activity against _Pseudomonas_ strains (MIC’s 8–64 µg/ml), but they were found not to protect mice against

infection by _Pseudomonas aeruginosa_ [15]. The structures of both families contain a 3′-deoxyuridine sugar attached via an 4′,5′-enamide linkage to the carboxyl group of an N-methyl

2,3-diaminobutyric acid (DABA) residue, to which amino acids are attached on both nitrogen substituents (see Fig. 2). To the α-amino group of DABA is attached either Met (mureidomycins) or

Ala (pacidamycins), which is in turn attached via a urea linkage to a C-terminal aromatic amino acid, either _meta_-tyrosine (mureidomycins), or Trp or Phe (pacidamycins). To the β-amino

group of the DABA residue is attached in most cases a _meta_-tyrosine residue, except in pacidamycin D, which contains Ala. Two further mureidomycins E and F were later reported, containing

a bicylic derivative of _meta-_tyrosine at the amino-terminal position [16], also found in the closely related napsamycins, which were reported in 1994 [17]. The sansanmycins were reported

in 2007, and contain the same structural skeleton as the mureidomycins, but contain Trp at the C-terminal position, and contain either Met, Leu, or methionine sulfoxide at position 4 [18,

19]. The sansanmycins showed antipseudomonal activity, but also showed activity against _Mycobacterium tuberculosis_ (MIC 8–20 µg/ml) [19]. The structures of these classes of uridyl peptide

antibiotics are shown in Fig. 2. A series of synthetic dihydropacidamycin analogues in which the 4′-5′ enamide was absent were prepared by Microcide Inc. The parent 4_R_-dihydropacidamycin

retained antipseudomonal activity, but with somewhat reduced MIC (64 µg/ml) compared with pacidamycin D [20]. Synthetic analogues containing Phe or Leu at position 4, and Trp or Tyr at

position 5, showed best antipseudomonal activity (MIC 4–16 µg/ml) [20]. An analogue containing 4-fluorophenylalanine in place of Met at position 4 showed antimicrobial activity against

clinical _E. coli_ strains (MIC 4–8 µg/ml), as well as _Mycobacterium tuberculosis_ (MIC 4–10 µg/ml) [21]. In 2011, Okamoto et al. published a total synthesis of pacidamycin D [22], which

they have used to synthesise further analogues varying the N-terminal dipeptide chain [23]. They have reported that meta-tyrosine in the amino-terminal position is considerably more active

than L-Tyr, and that the stereochemistry of the 2,3-DABA is important for both MraY inhibition and antimicrobial activity [23]. The anti-TB activity of the sansanmycin series has been

developed significantly by Tran et al., via chemical synthesis of a set of dihydrosansanmycin analogues [24]. They found that dihydrosansanmycin B had significantly improved anti-TB activity

(MIC50 0.3 µM) compared with sansanmycin B (MIC50 9.5 µM). Structure-activity studies revealed that analogues containing glycine at the N-terminal amino acid showed comparable activity

(MIC50 0.63 µM), and that a further modification of a cyclohexyl group at position 4 led to an analogue with MIC50 80 nM that was a potent MraY inhibitor (IC50 30 nM), as shown in Fig. 3

[24]. The biosynthetic gene cluster for the pacidamycin antibiotics, containing a number of non-ribosomal peptide synthetase genes, was identified in _Streptomyces coeruleorubidus_ in 2010

by Rackham et al. [25] and Zhang et al. [26]. The unusual ureidopeptide moiety at the carboxyl terminus of the peptide chain is assembled via carboxyl activation of Ala by PacN, followed by

carboxylation, and then peptide bond formation catalysed by ligase PacL [27]. The diamino acid DABA is biosynthesised from l-threonine by a pyridoxal-5′-phosphate-dependent β-replacement

reaction, also observed for mureidomycin biosynthesis in _Streptomyces flavidovirens_ [28], using l-aspartate as a nucleophile, followed by a β-elimination reaction [26]. The modified

uridine nucleoside is formed via oxidation of uridine to the 5′-aldehyde, followed by transamination to 5′-amino-uridine, followed dehydration of the 4′-hydroxyl group [29]. The crystal

structure of the novel dehydratase enzyme Pac13 has been determined, implicating His-42 in the catalytic mechanism [30]. The additional N-terminal Ala found in some pacidamycins and

mureidomycins is added by ligase PacB, that uses Ala-tRNA as an amino acid donor [31]. The unusual amino acid meta-tyrosine is biosynthesised from L-Phe by a novel non-haem iron- and

tetrahydrobiopterin-dependent hydroxylase [32]. Mutasynthesis has been used to generate novel chlorinated pacidamycin derivatives [33], and modified sansanmycins [34]. The modified

sansanmycins were reported to retain antimicrobial activity, in some cases with reduced activity, but MX-6 containing 4-fluorophenylalanine at the C-terminus showed enhanced antimicrobial

activity against _B. subtilis_ and _M. tuberculosis_ [34]. THE LIPOSIDOMYCIN GROUP OF LIPONUCLEOSIDE ANTIBIOTICS (LIPOSIDOMYCINS, CAPRAZAMYCINS) The liposidomycins are liponucleoside natural

products containing an aminoglycoside sugar, which were reported by Isono et al. in 1985 [1], and their molecular structures reported in 1988 [35]. They show antimicrobial activity against

_Mycobacterium_ strains (MIC 1.6 µg/ml) [1]. The caprazamycins were reported in 2003: they share the same structural skeleton as the liposidomycins, as shown in Fig. 4, but the

3-methylglutaryl substituent is glycosylated by an additional l-rhamnose sugar [36, 37]. Synthetic uridine-based analogues of the liposidomycins containing the aminoribofuranoside sugar

retain MraY inhibition activity (IC50 0.14–50 µM), but show weaker antimicrobial activity, demonstrating the importance of the lipophilic substituent, probably needed for cellular uptake

[38,39,40]. Fer et al. have published a further series of uridine-based analogues containing a lipophilic group linked via a triazole heterocycle, which show antimicrobial activity against

_Staphylococcus aureus_, and inhibit MraY with IC50 values in the range 100–1000 µM [41]. Ichikawa et al. have synthesised analogues of caprazamycin containing an alkyl chain in place of the

glutarate diester sidechain, which retain antimicrobial activity but show enhanced stability [42]. A semisynthetic caprazamycin derivative CPZEN-45 is active in animal models for protection

against tuberculosis infection, and Ichizaki et al. published in 2013 that CPZEN-45 inhibits transferase WecA in _M. tuberculosis_, involved in lipopolysaccharide biosynthesis, rather than

MraY [43]. The biosynthetic gene cluster for production of the caprazamycins in _Streptomyces_ sp. MK730-62F2 was identified in 2009 by Kaysser et al. [44]. The biosynthetic pathway involves

the formation of uridine 5′-aldehyde, followed by a pyridoxal-5′-phosphate-dependent reaction with glycine to form a uridine–amino acid adduct, followed by an

_S_-adenosylmethionine-dependent reaction transferring a 3-amino-3-carboxypropyl group [44]. A gene deletion strain, in which _cpz21_ encoding an acyltransferase enzyme acting late in the

biosynthetic pathway had been deleted, was found to accumulate the caprazamycin aglycone [44]. The genes responsible for addition of the l-rhamnose sugar found in caprazamycins have been

identified, allowing the heterologous gene expression of intact caprazamycins [45]. The biosynthetic pathway for the aminoribosyl sugar moiety found in the caprazamycin, muraymycin and other

nucleoside natural products has also been shown to proceed via uridine 5′-aldehyde, and was reported by van Lanen and co-workers [46,47,48]. THE MURAYMYCIN GROUP OF LIPO-UREIDYL PEPTIDE

NUCLEOSIDE ANTIBIOTICS The muraymycins were reported in 2002 by McDonald et al., isolated from a _Streptomyces sp_. strain [49]. Their structure contains an aminoribofuranoside

monosaccharide attached to the 5′-position of a uridine–amino acid, similar to that found in the liposidomycins and caprazamycins, as shown in Fig. 5, and a ureidopeptide structure linked

via a 3-aminopropyl moiety [49]. The muraymycins also target translocase MraY (IC50 0.027 µg/ml), show antimicrobial activity against strains of _Staphylococcus aureus_ (MIC 2–16 µg/ml) and

_Enterococcus_ (MIC 16–64 µg/ml), and were reported to protect mice against _S. aureus_ infection (ED50 1.1 mg/kg) [49]. Several bioactive muraymycin analogues have been generated via total

chemical synthesis. Tanino et al. have synthesised analogues in which the hydroxyleucine residue is replaced by an alkyl sidechain, which show MraY inhibition activity (IC50 0.33  µM), and

retain antimicrobial activity [50]. The same group have reported that the epicapreomycidine amino acid (a cyclic analogue of arginine) can be replaced by arginine, lysine or ornithine

residues, and that these synthetic analogues retain antimicrobial activity [51]. Takeoka et al. have prepared further analogues with l-arginine in place of epicapreomycidine, in which the

C-terminal amino acid is removed, which retain full MraY inhibition activity, and show enhanced antimicrobial activity against Pseudomonas strains [52]. Spork et al. have synthesised an

analogue of muraymycin lacking the aminoribose sugar which retains activity for MraY inhibition (IC50 2 µM) [53]. The ω-guanylated fatty acid, which is found in the most active muraymycins,

has been shown to assist localisation of the antibiotic into the cell membrane [54]. The total synthesis of muraymycin D1 was reported in 2016 by Mitachi et al. [55], enabling the synthesis

of further analogues. The biosynthetic gene cluster for the biosynthesis of muraymycin in _Streptomyces_ sp. NRRL 30471 was reported in 2011 [56]. THE CAPURAMYCIN GROUP OF CAPROLACTAM

NUCLEOSIDE ANTIBIOTICS (CAPURAMYCIN, A-500359A) Capuramycin, a nucleoside antibiotic produced by _Streptomyces griseus_, containing a uronic acid monosaccharide attached to the 5′ position

of a modified uridine nucleoside, to which is attached a seven-membered caprolactam ring, as shown in Fig. 6, was first reported in 1986 [57, 58]. Capuramycin and a methylated derivative

A-500359A, which shows antimicrobial activity against _Mycobacterium smegmatis_ (MIC 2–16 µg/ml) and potent MraY inhibition (IC50 0.017 µg/ml), was then reported in 2003 [59, 60]. A-500359E,

which lacks the aminocaprolactam ring, shows potent inhibition of MraY (IC50 0.027 µM), but lacks antimicrobial activity [61]. Semisynthetic derivatives of A-500359E have been reported, in

which the aminocaprolactam is replaced by synthetic arylamines, which show potent MraY inhibition (IC50 10–40 ng/ml), and antimicrobial activity against _Mycobacterium_ strains (MIC 0.5–2 

µg/ml) [62]. Acylation of capuramycin on the 2′ hydroxyl group gave a further series of bioactive derivatives, including a decanoyl derivative that shows very potent activity against _M.

tuberculosis_ (MIC 0.06 µg/ml) [63]. A biosynthetic gene cluster for a closely related capuramycin antibiotic A-503083B in _Streptomyces_ sp. SANK 62799 was reported in 2010 [64]. The

biosynthetic steps for attachment of a caprolactam moiety were elucidated, via carboxy methyltransferase CapS and transferase CapW [64]. The biosynthetic gene cluster for capuramycin

A-102395 has also been reported, involving the incorporation of l-threonine into uridine 5′-carboxamide [65]. Transferase CapW has been used to prepare of a set of 43 semisynthetic bioactive

capuramycin derivatives [66]. Several of these analogues retained similar antimicrobial activity to the parent compound, with three analogues showing enhanced activity against _M.

smegmatis_ and _M. tuberculosis_ [66]. COMPARISON OF ANTIMICROBIAL ACTIVITIES OF NUCLEOSIDE NATURAL PRODUCTS The nucleoside natural product antibiotics show very interesting and varied

antimicrobial activities. The mureidomycins show particularly potent antimicrobial activity against _Pseudomonas aeruginosa_ (MIC 0.1–3 µg/ml), a bacterium responsible for

antibiotic-resistant infections around the world, and can protect mice against infection by _Pseudomonas aeruginosa_ [11, 12]. The pacidamycins and napsamycins also show antipseudomonal

activity, but synthetic dihydropacidamycins containing modifications at position 4 (see Fig. 2) showed new antimicrobial spectrum against _Escherichia coli_ (MIC 4–8 µg/ml) and _Citrobacter

freundii_ (1.0 µg/ml) [20, 21]. Given that the MraY sequences from these organisms are quite closely related, it seems likely that these changes in antibacterial spectrum are caused by

changes in uptake. The liposidomycin and caprazamycin liponucleosides show activity against strains of _Mycobacterium_ (MIC 1.6 µg/ml) [2]. The synthetic caprazamycin derivative CPZEN-45 has

shown efficacy against both drug-sensitive and extremely drug-resistant Mtb in a mouse model of acute tuberculosis, and is in clinical trials against TB infection [43]. Capuramycins also

show potent activity against _Mycobacterium smegmatis_ (MIC 2–16 µg/ml) [59, 60], and semisynthetic derivatives show enhanced anti-Mtb activity [62, 63]. The activity of the sansanmycins

against _Mycobacterium tuberculosis_ has been greatly enhanced in synthetic dihydrosansanmycins containing modifications at position 4 (MIC50 0.04–0.6 µM) [24]. The muraymycin antibiotics

show antimicrobial activity against _Staphylococcus aureus_ (MIC 2–16 µg/ml) and _Enterococcus_ (MIC 16–64 µg/ml), and can protect mice against _S. aureus_ infection [49]. Synthetic

analogues containing two l-arginine residues show modified antimicrobial spectrum, notably against _Pseudomonas_ strains (MIC 4–8 µg/ml) [52]. MECHANISM OF INHIBITION OF TRANSLOCASE MRAY BY

NUCLEOSIDE ANTIBIOTICS KINETIC MECHANISM OF INHIBITION OF TRANSLOCASE MRAY Translocase MraY catalyses the first step of the lipid cycle of bacterial peptidoglycan biosynthesis, namely the

reaction of UDPMurNAc-L-Ala-γ-D-Glu-m-DAP-D-Ala-D-Ala (UDPMurNAc-pentapeptide) with lipid carrier undecaprenyl phosphate, to form lipid intermediate 1

(undecaprenyl-diphospho-MurNAc-pentapeptide), releasing uridine 5′-monophosphate [67]. Translocase MraY is an integral membrane protein, shown to contain ten transmembrane helices [68]. The

MraY-catalysed reaction is a phosphotransfer reaction, shown in Fig. 7, whose catalytic mechanism could either proceed via a single-step phosphotransfer, or a two-step mechanism involving an

active site nucleophile [67]. Three aspartic acid residues in _E. coli_ MraY (Asp-115, Asp-116, Asp-267), found on cytoplasmic loops, were shown to be essential for activity, and it has

been proposed that two Asp residues bind the active site Mg2+ cofactor, while the third may be a catalytic nucleophile [69]. Mureidomycin A has been found to act as a slow-binding inhibitor

(_K__i_ 35 nM, _K__i_* 2 nM) for solubilised _E. coli_ MraY, using a continuous fluorescence enhancement assay, showing competitive enzyme inhibition towards both UDPMurNAc-pentapeptide and

polyprenyl-phosphate substrates [70]. Liposidomycin B also acts as a slow-binding inhibitor of _E. coli_ MraY (_K__i_* 90 nM), showing non-competitive enzyme inhibition towards

UDPMurNAc-pentapeptide, but competitive inhibition towards dodecaprenyl phosphate [71]. By contrast, tunicamycin is a reversible inhibitor of _E. coli_ MraY (_K__i_ 0.6 µM) showing

competitive enzyme inhibition towards UDPMurNAc-pentapeptide, but non-competitive inhibition towards dodecaprenyl phosphate [71]. Muraymycin D2 and synthetic analogues thereof were found by

Tanino et al. to show competitive inhibition towards UDPMurNAc-pentapeptide (_K__i_ 7.6 nM), but non-competitive inhibition versus undecaprenyl phosphate, against _B. subtilis_ MraY using a

radiochemical assay [51]. Hence there are some differences in the kinetic mechanism of MraY inhibition shown by the different classes of nucleoside antibiotics. The possibility that the

mureidomycins might be mechanism-based inhibitors, reacting via the enamide functional group, which might be expected to be chemically reactive, has been found not to be the case, from

studies on model enamide-containing analogues [72], and since synthetic dihydropacidamycin and dihydrosansanmycin analogues retain MraY inhibition [20, 21, 24]. The observed slow-binding

inhibition is therefore probably due to a conformational change in the protein structure, discussed in section ‘Structure of _Aquifex aeolicus_ MraY and its complexes with nucleoside

antibiotics ‘. Studies on analogues of mureidomycins have found that the amino terminus of the peptide chain and the N-methyl amide group of DABA are both important for MraY inhibition [73,

74], leading to a proposal that the amino terminus might bind in place of the Mg2+ cofactor, positioned via a _cis_-amide rotamer in the peptide chain [74]. The amino group of the

aminoribofuranose monosaccharide of liposidomycins and caprazamycins is also known to be important for activity [39]. STRUCTURE OF _AQUIFEX AEOLICUS_ MRAY AND ITS COMPLEXES WITH NUCLEOSIDE

ANTIBIOTICS In 2013 the crystal structure of the _Aquifex aeolicus_ MraY was determined, confirming the arrangement of ten transmembrane α-helices [75]. The protein was found to crystallise

as a dimer, with transmembrane helix 9 strongly bent and protruding into the membrane. The active site contained the three catalytic Asp residues, close to the Mg2+ cofactor, with Asp-265

positioned closest to the Mg2+ ion [75]. There is a triad of three histidine residues (His-324, His-325, and His-326; HHH motif) conserved in bacterial sequences of the polyprenyl-phosphate

N-acetylhexosamine 1-phosphate transferase (PNPT) superfamily, which are positioned on loop E on the opposite side of the active site, 10–13 Å from the three catalytic Asp residues, shown in

Fig. 8. The structure of a complex of _A. aeolicus_ MraY with muraymycin D2 was published in 2016 [76]. Upon binding of muraymycin D2, transmembrane helix 9b (TM9b) moves away from the

active site and the HHH motif (conserved across the PNPT family) in loop E extends, which widens and reshapes the active site, hence there is a significant conformational change upon ligand

binding [76], which may be important for the catalytic cycle of MraY, and potentially could explain the slow-binding inhibition of MraY observed for some nucleoside natural product

inhibitors [70, 71]. Binding of muraymycin D2 to the MraY active site does not involve the catalytic Asp residues and does not require Mg2+, but several other binding interactions were

elucidated [76]. The uracil base is bound via π-π-stacking interactions to Phe-262 (see Fig. 9a), as well as hydrogen-bonding interactions to the uracil carbonyl and NH groups. The amino

group of the aminoribose moiety is bound by Asp-193, whose mutation greatly reduces affinity for muraymycin D2, and Asn-190. The strong binding of the amino group of the aminoribose helps to

rationalise why this group is important for activity in several nucleoside antibiotics [39, 73]. The carboxyl terminus of the peptide chain is bound by Gln-305, a residue that is conserved

in bacterial MraY homologues, and the epicapreomycidine amino acid is bound by His-324 and His-325 [76]. In 2017 the structure of a complex of _Clostridium boltae_ MraY with tunicamycin was

published [77]. The overall conformation of the protein was similar to the MraY–muraymycin complex, but some similarities and differences in the enzyme–ligand binding interactions were

observed [77, 78]. As in the MraY–muraymycin complex, the uracil base was bound in a small cavity, interacting via π-π-stacking interactions to Phe-228 (see Fig. 9b), and the 4-carbonyl

group binding to Asn-221. The HHH motif (His-290, His-291) are also involved in ligand binding, in this case to the GlcNAc 4′- and 6′-hydroxyl groups. However, unlike the MraY–muraymycin

complex, tunicamycin was found to interact with the catalytic Asp residues, with the tunicamine 9′-hydroxyl group interacting with Asp-231, but in the absence of Mg2+ [77]. The structure of

tunicamycin bound to its eukaryotic target enzyme GlcNAc-1-phosphate transferase involved in N-linked glycoprotein biosynthesis was also reported in 2018, showing some differences in active

site binding, compared to MraY [79]. Hence both nucleosides bind to an MraY structure that has undergone a conformational change, both show specific binding for the uracil base and some

involvement of the HHH motif in ligand binding, but muraymycin and tunicamycin show different polar contacts in the MraY active site [78]. INTERACTION WITH PROTEIN–PROTEIN INTERACTION SITE

FOR BACTERIOPHAGE LYSIS PROTEIN E _E. coli_ MraY is also targetted by an antibacterial lysis protein E from bacteriophage ϕX174, which interacts with Phe-288 and Glu-287 of MraY, on the

exterior face of transmembrane 9 of MraY, via an Arg-Trp-x-x-Trp sequence motif near the N-terminus of the E protein [80]. The presence of a guanidine-containing amino acid epicapreomycidine

in muraymycin, and two aromatic residues (Trp or m-Tyr and a second m-Tyr) in the mureidomycin/pacidamycin structures, is reminiscent of this Arg-Trp-x-x-Trp motif. Rodolis et al. have

shown that pacidamycin 1 and a synthetic muraymycin analogue showed significantly reduced activity against site-directed F288L and E287A MraY mutant enzymes [81], suggesting that parts of

the antibiotic structure somehow aid the targetting of MraY in vivo, perhaps aiding uptake into the cell via a hydrophobic channel present in the structure of MraY [75], as shown in Fig. 10.

This hypothesis might explain how these agents of molecular weight 600–1200 Da are able to access the MraY active site on the inner face of the cytoplasmic membrane, and also why it has

proved difficult to design small analogues of these nucleoside antibiotics that retain both MraY inhibition and antimicrobial activity. INHIBITION OF OTHER BACTERIAL PHOSPHO-NUCLEOTIDE

TRANSFERASE ENZYMES BY NUCLEOSIDE NATURAL PRODUCT ANALOGUES There are homologues of MraY involved in lipid-linked cycles responsible for the biosynthesis of enterobacterial common antigen

and O-antigen lipopolysaccharide in Gram-negative bacteria (WecA) [82], and teichoic acid in Gram-positive bacteria (TagO), both of which are integral membrane proteins that utilise

UDP-GlcNAc and undecaprenyl phosphate as substrates [83]. CPZEN-45, a semisynthetic caprazamycin derivative undergoing clinical trials for treatment of tuberculosis, was found to inhibit

transferase WecA in _M. tuberculosis_ >20-fold more strongly than MraY, and CPZEN-45 also inhibits _B. subtilis_ TagO 8-fold more tightly than _B. subtilis_ MraY [43]. Selective synthetic

inhibitors for TagO have also been published, based on the existing drug ticlopidine [84]. In contrast, the muraymycin analogues prepared by Tanino et al. were highly selective for MraY

inhibition over _E. coli_ WecA [51], as were the dihydrosansanmycin analogues of Tan et al. [24]. A homologue of WecA (GacO) has been found to catalyse the formation of

undecaprenyl-diphospho-GlcNAc in the biosynthesis of the Lancefield group A carbohydrate in _Streptococcus pyogenes_ [85]. A further group of small 20–25 kDa phospho-sugar transferase

enzymes utilising a UDP-di-_N_-acetyl-bacillosamine substrate are involved in _N_-glycoconjugate biosynthesis in _Campylobacter jejuni_ [86]. Synthetic peptidyl-uridine inhibitors have been

synthesised as inhibitors of _C. jejuni_ PglC, with IC50 values in the range 40–250 µM [87]. In conclusion, nature produces several classes of uridine-based nucleoside antibiotics that

target translocase MraY in the bacterial peptidoglycan biosynthetic pathway. Total synthesis of modified nucleoside analogues, and exploitation of the biosynthetic machinery for these

natural products, offer considerable promise for the development of highly active antimicrobial agents to treat antibiotic-resistant infections. Understanding of the MraY structure will aid

the development of selective MraY inhibitors, and the discovery of MraY homologues such as WecA and TagO has identified further interesting targets for antibacterial drug discovery by

nucleoside analogues. NOTE ADDED IN PROOF A further protein crystallography study of _A. aeolicus_ MraY has very recently been published, describing the structures of MraY complexes with

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nucleoside natural products. Nat. Commun. 2019;10:2917. Download references ACKNOWLEDGEMENTS Research in the author’s laboratory was supported by an EPSRC CASE PhD studentship (to RVK) with

LifeArc Ltd. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK Timothy D. H. Bugg & Rachel V. Kerr Authors * Timothy D.

H. Bugg View author publications You can also search for this author inPubMed Google Scholar * Rachel V. Kerr View author publications You can also search for this author inPubMed Google

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commemorating Dr Kiyoshi Isono and his important contributions to the study of nucleoside antibiotics. Dr Isono led the discovery of the liposidomycin natural products in 1985, one of the

first studies in this field, which established that nucleoside antibiotics could be selective antibacterial agents. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE

THIS ARTICLE Bugg, T.D.H., Kerr, R.V. Mechanism of action of nucleoside antibacterial natural product antibiotics. _J Antibiot_ 72, 865–876 (2019). https://doi.org/10.1038/s41429-019-0227-3

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