Biosynthetic genes for aminoglycoside antibiotics

Kudo, Fumitaka; Eguchi, Tadashi

doi:10.1038/ja.2009.76

Cited by 87 publications

(88 citation statements)

References 45 publications

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“…To examine how BtrN employs AdoMet radical chemistry, we have determined its structure with AdoMet and substrate to 1.56 Å resolution. We find a previously undescribed modification to the core AdoMet radical fold: instead of the canonical (β/α) 6 architecture, BtrN displays a (β 5 /α 4 ) motif. We further find that an auxiliary [4Fe-4S] cluster in BtrN, thought to bind substrate, is instead implicated in substrate-radical oxidation.…”

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confidence: 99%

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Goldman

Grove²,

Booker

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The 2-deoxy-scyllo-inosamine (DOIA) dehydrogenases are key enzymes in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics. In contrast to most DOIA dehydrogenases, which are NAD-dependent, the DOIA dehydrogenase from Bacillus circulans (BtrN) is an S-adenosyl-L-methionine (AdoMet) radical enzyme. To examine how BtrN employs AdoMet radical chemistry, we have determined its structure with AdoMet and substrate to 1.56 Å resolution. We find a previously undescribed modification to the core AdoMet radical fold: instead of the canonical (β/α) 6 architecture, BtrN displays a (β 5 /α 4 ) motif. We further find that an auxiliary [4Fe-4S] cluster in BtrN, thought to bind substrate, is instead implicated in substrate-radical oxidation. High structural homology in the auxiliary cluster binding region between BtrN, fellow AdoMet radical dehydrogenase anSME, and molybdenum cofactor biosynthetic enzyme MoaA provides support for the establishment of an AdoMet radical structural motif that is likely common to ∼6,400 uncharacterized AdoMet radical enzymes.radical SAM enzyme | iron-sulfur cluster fold | twitch domain D ue to mounting antibiotic resistance, the discovery and/or modification of antibiotic compounds to fight bacterial infection is a vital task of our time (1). Understanding antibiotic biosynthesis is an important first step in being able to engineer a new wave of therapeutic molecules. Aminoglycosides are a class of antibiotics that inhibit protein synthesis by interacting with the 30S subunit of the bacterial ribosome and have had considerable success in the clinical setting (2). Most FDA-approved aminoglycosides, including neomycin, gentamicin, and kanamycin, share a common glucose-6-phosphate-derived 2-deoxystreptamine (DOS) structural core (3) (blue in Scheme 1). Although the majority of aminoglycoside-producing bacteria use similar reaction sequences to biosynthesize this DOS core, including the penultimate two-electron oxidation of 2-deoxy-scyllo-inosamine (DOIA) to amino-dideoxy-scyllo-inosose (amino-DOI) (Scheme 1, A), identification of the enzymes responsible has not always been straightforward. For example, the btrE gene product in the butirosin B producing Bacillus circulans was proposed to be an NADdependent enzyme responsible for the generation of amino-DOI, as in other aminoglycoside pathways (4). This hypothesis proved incorrect upon further sequence and biochemical analysis (5, 6). Instead, Yokoyama et al. found that production of amino-DOI required another enzyme, BtrN, an S-adenosyl-L-methionine (AdoMet, SAM) radical dehydrogenase (5). Here, we report structures of catalytic and noncatalytic forms of BtrN, providing a structural basis for the unique reaction it performs.The AdoMet radical enzyme family catalyzes a diverse array of radical-based reactions, including sulfur insertions, complex chemical transformations and rearrangements, DNA and RNA modifications, and, in the case of BtrN, dehydrogenation (7). BtrN is one of two known members of the dehydrogenase subfamily of ...

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confidence: 99%

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Goldman

Grove²,

Booker

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…6,7 To prevent confusion, herein we use the btr gene-based names, which were used systematically by the Piepersberg group and also in our recent review. 8,9 The entire kanamycin biosynthetic gene cluster was expressed in Streptomyces venezuelae and the resultant recombinant strain was reported to produce kanamycin A, which indicates that the kan genes are responsible for the biosynthesis of kanamycins. 10 The kanC, kanS1, kanE, kanM1 and kanN genes expressing Streptomyces lividans reportedly produce paromamine, confirming that these genes, respectively, encode 2DOI synthase, Gln:2DOI aminotransferase, 2-deoxy-scyllo-inosamine dehydrogenase, UDP-GlcNAc:2DOS N-acetylglucosaminyltransferase and N-acetylparomamine deacetylase ( Figure 1).…”

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confidence: 99%

“…9 Alternatively, this set of enzymes might be responsible for the formation of NDP-3-amino-3-deoxyglucose (kanosamine), which is another possible glycosyl donor in the KanM2 reaction, to afford kanamycin C. 11 KanS2 is a protein that is homologous to KanS1 (37% identity/51% positive), which is involved in the double transamination in 2DOS biosynthesis. The difference in substrate specificity of these aminotransferases is an interesting issue to be resolved.…”

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Enzymatic activity of a glycosyltransferase KanM2 encoded in the kanamycin biosynthetic gene cluster

2009

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Kanamycin, discovered by Umezawa in 1957, is the most well-known 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotic. 1 Three structurally related kanamycins were identified from the producer Streptomyces kanamyceticus (Figure 1). Extensive biosynthetic studies of this class of aminoglycoside antibiotics, especially butirosin and neomycin, clarified that a unique aminocyclitol, 2DOS, is biosynthesized from D-glucose 6-phosphate by three crucial enzymes, 2-deoxy-scyllo-inosose (2DOI) synthase, L-glutamine (Gln):2DOI aminotransferase and 2-deoxy-scyllo-inosamine dehydrogenase. 2,3 After the 2DOS formation, uridine 5¢-diphospho-N-acetylglucosamine (UDP-GlcNAc):2DOS N-acetylglucosaminyltransferase catalyzes the glycosylation of 2DOS to give N-acetylparomamine, which is then deacetylated by N-acetylparomamine deacetylase to yield paromamine. 4 Paromamine is believed to be a branching biosynthetic intermediate, which is converted to either 4,6-disubstituted 2DOS aminoglycosides, such as kanamycin and gentamicin, or 4,5-disubstituted 2DOS aminoglycosides, such as neomycin and butirosin. Therefore, the regio-specificities and substrate specificities of glycosyltransferases that recognize paromamine as a glycosyl acceptor determine the core structures of aminoglycoside antibiotics.The biosynthetic gene cluster for kanamycin, comprising 24 open reading frames, was identified in 2004. 5 Two other research groups also deposited independently identical kanamycin biosynthetic genes by different symbols in the public DNA databases simultaneously. 6,7 To prevent confusion, herein we use the btr gene-based names, which were used systematically by the Piepersberg group and also in our recent review. 8,9 The entire kanamycin biosynthetic gene cluster was expressed in Streptomyces venezuelae and the resultant recombinant strain was reported to produce kanamycin A, which indicates that the kan genes are responsible for the biosynthesis of kanamycins. 10 The kanC, kanS1, kanE, kanM1 and kanN genes expressing Streptomyces lividans reportedly produce paromamine, confirming that these genes, respectively, encode 2DOI synthase, Gln:2DOI aminotransferase, 2-deoxy-scyllo-inosamine dehydrogenase, UDP-GlcNAc:2DOS N-acetylglucosaminyltransferase and N-acetylparomamine deacetylase (Figure 1). 11 Among these, the kanC gene was expressed in Escherichia coli and the recombinant KanC protein was confirmed to be 2DOI synthase. 6 Recombinant KanM1 protein was also reported to have weak glycosyltransferase activity with 2DOS as a glycosyl acceptor, and TDP-glucose or GDP-mannose as a glycosyl donor. 12 However, this KanM1 activity is contradictory to the presumed enzymatic function as suggested by the above-mentioned heterologous expression of UDP-GlcNAc:2DOS N-acetylglucosaminyltransferase in S. lividans. Detailed enzymatic analysis is thus necessary to elucidate the substrate specificity of KanM1 on using a pure enzyme.In this study, we heterologously expressed another glycosyltransferase gene, kanM2, in the kan gene cluster and investig...

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“…5 (As the biosynthetic gene cluster for neomycin was deposited with different symbols in DNA databases by multiple groups, we here use the butirosin biosynthetic btr gene-based code names, which are systematically utilized by the Piepersberg et al 6 and also in our recent review. 7 ) NeoB has also been reported to catalyze two transaminations: the conversion of neomycin C to 6¢¢¢-oxoneomycin C, which is a reverse reaction of the postulated in vivo biosynthetic reaction (Figure 1), and the conversion of 6¢-oxoparo- Figure 2 NMR spectra of neomycin C in D 2 O synthesized with biosynthetic enzymes. (a) 1 H-NMR (500 MHz) and (b) 13 C-NMR (125 MHz) were recorded on DRX500 (Bruker, Rheinstetten, Germany).…”

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“…A comparison of aminoglycoside biosynthetic gene clusters indicated the involvement of NeoN, a putative radical SAM enzyme that is also encoded in the paromomycin and lividomycin biosynthetic clusters. 5,7 As such an epimerization by a radical SAM enzyme is unprecedented 10 and would be an important tool to synthesize structurally diverse aminoglycosides, the functional characterization of NeoN is important and is currently underway.…”

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Enzymatic preparation of neomycin C from ribostamycin

et al. 2009

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The high regio-and stereospecificities of enzymes and their ability to catalyze reactions in the absence of organic solvents make enzymatic synthesis of antibiotics an attractive way to prepare complex natural products. Enzymatic total syntheses of several complex natural products have been recently achieved, which illustrates the power of this synthetic methodology. 1,2 Combinations of biosynthetic enzymes and substrates, including unnatural types, can afford structurally diverse natural product-like compounds. 3 In this study, we report the enzymatic synthesis of the aminoglycoside antibiotic neomycin C from ribostamycin by the use of four neomycin biosynthetic enzymes. So far, all nine enzymes for ribostamycin biosynthesis from D-glucose 6-phosphate have been characterized using the enzymes derived from the butirosin producer Bacillus circulans and the neomycin producer Streptomyces fradiae. 4 Furthermore, two neomycin biosynthetic enzymes NeoF and NeoD were recently found to catalyze the glycosylation of ribostamycin using Figure 1 Biosynthesis of neomycin C from ribostamycin.

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Biosynthetic genes for aminoglycoside antibiotics

Cited by 87 publications

References 45 publications

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Enzymatic activity of a glycosyltransferase KanM2 encoded in the kanamycin biosynthetic gene cluster

Enzymatic preparation of neomycin C from ribostamycin

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