Biosynthesis of 2-Deoxystreptamine by Three Crucial Enzymes in Streptomyces fradiae NBRC 12773

Kudo, Fumitaka; Yamamoto, Yoh; Yokoyama, Kenichi; Eguchi, Tadashi; Kakinuma, Katsumi

doi:10.1038/ja.2005.104

Cited by 45 publications

(50 citation statements)

References 32 publications

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“…Next, an NADdependent dehydrogenase catalyzes the dehydrogenation at C-1 of 2DOIA to give 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). 26 Amino-DOI is then converted to 2DOS by dual functional Gln:2DOI aminotransferase to complete the 2DOS biosynthesis. 27 Then, 2DOS is glycosylated using glycosyltransferase with UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) as a glycosyl donor to give N-acetylparomamine, whose N-acetyl group is removed using a deacetylase to give paromamine.…”

Section: Neomycin-related Biosynthetic Genesmentioning

confidence: 99%

“…For example, BtrE encoded in the butirosin gene cluster was presumed to be responsible for the dehydrogenation of 2DOIA in the 2DOS pathway, although it showed no expected activity. 26 A butirosin-specific radical SAM enzyme, BtrN, was then found to be responsible for dehydrogenation instead of BtrE. 33 In addition, it is noteworthy that C-6¢ of paromomycin and lividomycin remains in the hydroxy group, even though a set of dehydrogenase and aminotransferase responsible for the neamine biosynthesis is conserved.…”

Section: Neomycin-related Biosynthetic Genesmentioning

confidence: 99%

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Biosynthetic genes for aminoglycoside antibiotics

Kudo

Eguchi

2009

J Antibiot

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Biosynthetic studies of aminoglycoside antibiotics have progressed remarkably during the last decade. Many biosynthetic gene clusters for aminoglycoside antibiotics including streptomycin, kanamycin, butirosin, neomycin and gentamicin have been identified to date. In addition, most butirosin and neomycin biosynthetic enzymes have been functionally characterized using recombinant proteins. Herein, we reanalyze biosynthetic genes for structurally related 2-deoxystreptamine (2DOS)-containing aminoglycosides, such as kanamycin, gentamicin and istamycin, based on genetic information including characterized biosynthetic enzymes in neomycin and butirosin biosynthetic pathways. These proposed enzymatic functions for uncharacterized enzymes are expected to support investigation of the complex biosynthetic pathways for this important class of antibiotics.

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Section: Neomycin-related Biosynthetic Genesmentioning

confidence: 99%

Section: Neomycin-related Biosynthetic Genesmentioning

confidence: 99%

Biosynthetic genes for aminoglycoside antibiotics

Kudo

Eguchi

2009

J Antibiot

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“…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).…”

mentioning

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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“…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.…”

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confidence: 99%

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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Biosynthesis of 2-Deoxystreptamine by Three Crucial Enzymes in Streptomyces fradiae NBRC 12773

Cited by 45 publications

References 32 publications

Biosynthetic genes for aminoglycoside antibiotics

Biosynthetic genes for aminoglycoside antibiotics

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

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